Labeling of antibodies

ABSTRACT

Provided herein are methods for producing site specific PEG modifications to single domain antibodies (e.g., VHHs). Methods for producing site-specific ally conjugated bivalent single domain antibodies (e.g., VHHs) are also provided. Methods for labeling (e.g., with a fluorophore or radionuclide) site-specifically PEGylated single domain antibodies and site-specifically conjugated bivalent single domain antibodies are also provided.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofinternational PCT application, PCT/US2016/055074, filed Oct. 1, 2016,which claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication, U.S. Ser. No. 62/236,117, filed on Oct. 1, 2015 each ofwhich is incorporated by reference herein.

GOVERNMENT SUPPORT

This invention was made with government support under AI087879-06,GM106409-03, and GM 100518-04 awarded by National Institutes of Health.The government has certain rights in the invention.

BACKGROUND

Positron emission tomography (PET) is a powerful technology for medicaland biological imaging and the scope of PET applications is expandingrapidly. The development of suitable PET tracers is critical to PETtechnology. Accordingly new PET tracers, such as peptide/protein basedtracers would be useful to specifically label tissues in therapeuticand/or diagnostic applications.

SUMMARY

Some aspects of the disclosure are based on the recognition thatantibody binding fragments, and in particular the single domain antibodyfragment VHH, show an improved ability to detect antigens in vivo whensite specifically conjugated to a hydrophilic polymer (e.g., PEG) and aradiolabel (e.g. ⁸⁹Zr). Such antibodies are useful for inter aliadetecting tumors and lymphocyte infiltration into tumors in vivo.Accordingly, the present disclosure provides antibodies that are sitespecifically labeled with a hydrophilic polymer and/or a radiolabel, aswell as methods of making and using such antibodies.

The success of immunotherapies such as the administration of monoclonalantibodies against immune checkpoint inhibitors (e.g., CTLA4, PD-1 on Tcells, and PD-L1) warrants the development of new methods, systems, andcompositions for assessing whether an immunotherapy would be beneficialfor treating patients. As one example, macrophages can affect tumorgrowth by establishing either a detrimental or favorablemicroenvironment. Thus, the ability to image the present of myeloidcells is of diagnostic and therapeutic relevance and, compared totumor-specific markers^([1]), may be a more generally applicableapproach for detection of tumor cells^([2,3]). Immune cells often invadeor surround solid tumors^([1]). The success of immunotherapies such asthe administration of monoclonal antibodies against immune checkpointinhibitors (e.g., CTLA4, PD-1 on T cells, and PD-L1) may be assessed byimaging the presence and/or distribution of immune cells (e.g., myeloidand/or lymphoid cells) in a subject, for example, in a tumor of thesubject. Accordingly, new methods and compositions to explore and assessthe microenvironment of tumors non-invasively are needed.

Immune cells often infiltrate tumors and can thus serve as a proxy toassess the presence of a malignancy. The ability to imagetumor-infiltrating cells depends on the availability of targetingmolecules with the requisite affinity and specificity. Camelid-derivedsingle domain antibody fragments (VHHs) Provided herein is the designand synthesis of site-specifically PEGylated VHHs and engineeredbivalent VHHs fused site-specifically via their C-termini. VHHs, whichrecognize Class II MHC products and CD11b, were used to image immunecells, both in vitro and in vivo, by FACS, two-photon microscopy andpositron emission tomography (PET). Both PEGylated and bivalent VHHsstained lymphoid organs and stained them more efficiently than theirmonovalent non-PEGylated counterparts. By PET imaging, modified VHHsdetected engrafted melanoma and pancreatic tumors—as small as ˜1 mm insize—by virtue of their association with myeloid cells. Modified VHHsshowed significant improvement in fluorescence imaging, especially forthe PEGylated VHHs.

In one aspect, a chemical approach was chosen to link two fullyfunctional and properly refolded VHHs via their C-termini to ensure thattheir antigen binding capacity would not be compromised by modificationof one of the N-termini, and that the two binding sites thus createdwould be equivalent, in the manner of a full-sized antibody.Accordingly, bivalent VHHs were produced through application ofsortase-mediated enzymatic transformations in combination with clickchemistry. Mono- and bivalent VHHs were compared in in vitro (FACS)experiments, by two-photon microscopy on lymph nodes and spleen excisedfrom mice injected with VHHs, and by non-invasive in vivo immuno-PETimaging.

One minimally-invasive clinical diagnostic approach is the use of¹⁸F-2-fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET),which distinguishes areas of high metabolic activity, such as tumors,from surrounding tissue with lesser glucose uptake^([4]). These methodsmay not typically provide information on immune cells in the tumormicroenvironment. They can be used to track immune cells, usingisotopically labeled anti-CD11b, anti-Class II MHC, and anti CD8antibody fragments^([5-7]).

The comparatively large size of intact full-sized antibodies results ina long circulatory half-life and may also hinder efficient tissuepenetration^([8]). These considerations have driven the search forsmaller antibody-derived formats as alternative imaging tools^([1, 6]).Thus, provided herein are camelid single domain antibody fragments(VHHs), as the smallest antigen binding derivatives obtainable fromnaturally occurring antibodies[^(9]). VHHs may be enzymatically modifiedand have been used in a variety of applications includingimaging^([10]).

The production of bivalent single domain antibodies based on theirmonovalent equivalents could address issues of avidity, while retainingdesirable properties such as small size. For example, the bivalentderivatives of single domain antibodies maybe small enough to penetratetissues, be rapidly cleared from the circulation, yet benefit fromincreased avidity. On the other hand, tuning circulatory half-life couldimprove the staining efficiency of the targets by VHHs. Attachment ofsmall PEG groups (e.g., PEGylation) or other hydrophilic polymers couldbe used as a tool to tune persistence of a VHH in the circulation.Moreover, PEGylation can decrease the immunogenicity of VHHs, which isimportant in cases when repeated administration is required but in rarecases even the PEG functionality itself may be immunogenic^([12]). Thus,provided herein are methods for introducing site specific hydrophilicpolymer (e.g., PEG) and radiolabel (e.g., ⁸⁹Zr) modifications ontosingle domain antibodies (e.g., VHHs) and methods for producingsite-specifically conjugated bivalent single domain antibodies. In someembodiments, the single domain antibodies provided herein are sitespecifically modified with PEG and a ⁸⁹Zr. However, it should beappreciated that additional hydrophilic polymers and other radiolabelsmay be used to modify the antibodies (e.g., single domain antibodies)provided herein.

The details of certain embodiments of the invention are set forth in theDetailed Description of Certain Embodiments, as described below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe Definitions, Examples, Figures, and Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which constitute a part of thisspecification, illustrate several exemplary embodiments of the inventionand together with the description, serve to explain the principles ofthe invention. The embodiments disclosed in the drawings are exemplaryand do not limit the scope of this disclosure.

FIGS. 1A-1B show site-specific labeling of single domain antibodies(VHHs) using sortase and design (FIG. 1A) as well as (FIG. 1B)structures of the substrates. LPETG-GH₆ is SEQ ID NO: 5; GGH₆ is SEQ IDNO: 6; LPET is SEQ ID NO: 7; LPET-G₃ is SEQ ID NO: 8.

FIGS. 2A-2F show synthesis of VHH-derivatives (FIG. 2A and FIG. 2B).Characterizations were performed on all the products (FIG. 2C and FIG.2D). Here is shown the LC-MS and SDS-PAGE analysis for DC13. Numbersindicate the followings: #1: marker, #2: DC13, #3: DC13-DBCO, #4:DC13-azide-Texas Red, #5: DC13-dimer-Texas Red, #6: DC13-azide-Alexa647,#7: DC13-dimer-Alexa647. (FIGS. 2E-2F) PEGylation of VHHs andcharacterization. Numbers indicate the following: #1: marker, #2:DC8-alexa647-azide, #3: DC8-alexa647-PEG-5 kDa, #4: DC8-alexa647-PEG-10kDa, #5: DC8-alexa647-PEG-20 kDa. LPETG-XX is SEQ ID NO: 9; LPET-G₃ isSEQ ID NO: 8; LPET-G₃-Cys is SEQ ID NO: 10.

FIGS. 3A-3D (FIGS. 3A-3B) show dimers that are able to stain theirtargets with increased efficiency compared to their correspondingmonomers; for FIG. 3A: cells were stained in vitro with the indicatedconcentrations of Alexa647-labeled VHH monomers or dimers; for FIG. 3B:equal amount of DC8 monomer and DC8-dimer is injected IV. 2 h p.i., themice are euthanized and spleens are excised for two-photon imaging.(FIG. 3C) Equal amount of DC8 with or without PEG, both equipped withTexas Red is injected IV. 3 h p.i., the mice are euthanized and spleensare excised for two-photon imaging. (FIG. 3D) Equal amount of DC8 withor without PEG (0.4 nmols), all equipped with Alexa647 is injected IV. 3h p.i., the mice are euthanized and spleen is excised. Cells wereharvested and analyzed with FACS.

FIGS. 4A-4I (FIG. 4A) show synthesis of ¹⁸F-Tetrazine. (FIG. 4B)¹⁸F-labeling of VHH-dimers, monomers or PEG-VHHs. (FIG. 4C) radio-TLCanalysis of the ¹⁸F-labeling reactions. (FIGS. 4D-4I) ¹⁸F-DC8-dimer and18F-DC13-dimer detects lymphoid organs and reveals tumor-infiltratedimmune cells. PET images are represented in both coronal and sagittalviews for better visualization. CT images are provided for bettervisualization (FIG. 4H). (FIGS. 4D-4E) PET images of WT (FIG. 4D) andclass II MHC−/− (FIG. 4E) mice 2 h p.i. of ¹⁸F-DC8 dimer; lettersindicate: CV: cervical lymph nodes; BM: bone marrow; GB: gallbladder;LV: liver; KD: kidneys; Ints: intestines; BL: bladder; SP: spinal cord.(FIGS. 4F-4G) PET images of WT (FIG. 4F) and CD11b−/− (FIG. 4G) mice 2 hp.i. of ¹⁸F-DC13 dimer. (FIG. 4I) Tumor-associated class II MHC+ cells(FIG. 4I (coronal)) or CD11b+ cells (FIG. 4I (sagittal)) were visualizedusing ¹⁸F-DC13 dimer (FIG. 4I (coronal)) or ¹⁸F-DC8 dimer (FIG. 4I(sagittal)). Arrows are pointing at the tumors. In (FIG. 4I (sagittal))stars are showing the lymph nodes. PET scale bars have arbitrary units.Images are representative of two to four mice with similar results.

FIGS. 5A-5D (FIGS. 5A-5B) show postmortem biodistribution of ¹⁸F-VHHs inall organs. Mice were injected with same amount of either ¹⁸F-VHH or¹⁸F-VHH dimer. 3 h p.i., mice were euthanized and activity in differentorgans were measured. (FIG. 5C) VHH monomer can block binding of ¹⁸F-VHHdimer onto the lymphoid organs. WT mice were injected with differentamount of unlabeled DC8. ¹⁸F-DC8 dimer was injected 20 min later. PETSUVs are calculated based on PET images acquired 2 h p.i. of the¹⁸F-labeled DC8 dimer. (FIG. 5D) WT mice were injected with same amountof either ¹⁸F-DC8 with or without labeled-PEGs. 3 h p.i., the mice wereeuthanized and activity in different organs were measured.

FIGS. 6A-6H show LC-MS analysis of VHHs, their corresponding sortaggedproducts and dimers. DC8 and its derivatives (FIGS. 6A-6H).

FIGS. 7A-7H show LC-MS analysis of VHHs, their corresponding sortaggedproducts and dimers. DC13 and its derivatives (FIGS. 7A-7H).

FIG. 8 Shows a table of radiochemical yield (non-decay corrected) and aradio HPLC chromatogram. The column was luna 5u C18 100 Å 250×4.6 mm,the mobile phase was 60% CH3CN, 40% 0.1 M NH4.HCO2(aq), and the flowrate was 1 mL/min.

FIG. 9 Shows a schematic of the GE TRACERlab™ FXFN radiosynthesis moduleautomated synthesis manifold for [¹⁸F]-5.

FIG. 10 shows a semi-preparative HPLC trace of a typical radiosynthesisof [¹⁸F]-5.

FIG. 11 shows the PD-10 cartridge was eluted with PBS (10×500 μL), andeach fragment was collected into a new 1.5 mL tube. The desired product[¹⁸F]-VHHs usually eluted at tubes #4-7. Characterization (using[¹⁸F]-DC8-dimer as an example): rTLC chromatography (left panel[18F]-Tz5; right panel[¹⁸F]-DC8-dimer; at 20 min).

FIG. 12 shows fragments collection through PD-10 cartridge. Aftersize-exclusion chromatography, a 47±9% (n=5, non-decay corrected)radiochemical yield was obtained.

FIGS. 13A-13B show NMR spectra of [¹⁹F]tetrazine 5: ¹H-NMR (FIG. 13A)and ¹³C-NMR (FIG. 13B).

FIGS. 14A-14E show a schematic of a camelid heavy chain only antibodyand a conventional IgG. A VHH portion is shown within the circleindicated by the arrow (FIG. 14A). FIG. 14B is a schematicrepresentation of site-specific labeling of VHHs using sortase. LPETG-XXis SEQ ID NO: 9.

FIGS. 15A-15B shows the structure of the synthesized bi-orthogonalsortase substrate (FIG. 15A). The azide functionality allows installmentof PEG groups, and DFO chelator is used to install radiometal ⁸⁹Zrallowing PET imaging. FIG. 15B is a schematic representation ofpreparing PEGylated ⁸⁹Zr-labeled VHHs for PET imaging.

FIGS. 16A-16B shows that ⁸⁹Zr-PEGylated-VHH7 detects secondary lymphoidorgans and B16 tumor in a wild-type B6 mouse injected with B16 tumorcells (FIG. 16A), and ⁸⁹Zr-PEGylated-VHH7 detects secondary lymphoidorgans a wild-type B6 mouse not injected with B16 tumor cells (FIG.16B).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Antibodies are currently the fastest growing class of therapeutics.Although naked antibodies have proven their value as successfulpharmaceuticals, they suffer from some limitations such as low tissuepenetration and a long circulatory half-life. They have been conjugatedto toxic payloads, PEGs or other polymers, or radioisotopes to increaseand optimize their therapeutic efficacy. Although non-specificconjugation is suitable for most in vitro applications, for in vivoapplications, site-specifically modified antibodies may have advantages.Provided herein is a novel approach in which an antibody fragment istagged with two handles: one is used for the introduction of afluorophore or radio-isotope, and the second one is used to furthermodify the fragment with functionalities including a PEG moiety or asecond antibody fragment. Such modifications may improve desiredproperties (e.g., circulatory half-life or avidity). Exemplaryantibodies (e.g., single domain antibodies) provided herein, whichrecognize epitopes, such as Class II MHC, and CD11b showed high avidityand specificity. In some embodiments, antibodies conjugated to ahydrophilic polymer (e.g., PEG), and/or a fluorophore are referred to asconstructs. Such constructs were used to image cancers and could detecttumors as small as about 1 mm in size.

The term “antibody”, as used herein, refers to a protein belonging tothe immunoglobulin superfamily. The terms antibody and immunoglobulinare used interchangeably. The term “antibody” encompasses not onlyintact (e.g., full-length) polyclonal or monoclonal antibodies, but alsoantigen-binding fragments thereof (such as Fab, Fab′, F(ab′)2, Fv),single chain (scFv), mutants thereof, fusion proteins comprising anantibody portion, humanized antibodies, chimeric antibodies, diabodies,linear antibodies, single chain antibodies, multispecific antibodies(e.g., bispecific antibodies) and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen recognition siteof the required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. With some exceptions, mammalian antibodies aretypically made of basic structural units each with two large heavychains and two small light chains. There are several different types ofantibody heavy chains, and several different kinds of antibodies, whichare grouped into different isotypes based on which heavy chain theypossess. Five different antibody isotypes are known in mammals, IgG,IgA, IgE, IgD, and IgM, which perform different roles, and help directthe appropriate immune response for each different type of foreignobject they encounter. In some embodiments, an antibody is an IgGantibody, e.g., an antibody of the IgG1, 2, 3, or 4 human subclass.Antibodies from mammalian species (e.g., human, mouse, rat, goat, pig,horse, cattle, camel) are within the scope of the term, as areantibodies from non-mammalian species (e.g., from birds, reptiles,amphibia) are also within the scope of the term, e.g., IgY antibodies.

Only part of an antibody is involved in the binding of the antigen, andantigen-binding antibody fragments, their preparation and use, are wellknown to those of skill in the art. As is well-known in the art, only asmall portion of an antibody molecule, the paratope, is involved in thebinding of the antibody to its epitope (see, in general, Clark, W. R.(1986) The Experimental Foundations of Modern Immunology Wiley & Sons,Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed.,Blackwell Scientific Publications, Oxford). Suitable antibodies andantibody fragments for use in the context of some embodiments of thepresent invention include, for example, human antibodies, humanizedantibodies, domain antibodies, F(ab′), F(ab′)₂, Fab, Fv, Fc, and Fdfragments, antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; antibodies in which the FR and/or CDR1 and/orCDR2 and/or light chain CDR3 regions have been replaced by homologoushuman or non-human sequences; antibodies in which the FR and/or CDR1and/or CDR2 and/or light chain CDR3 regions have been replaced byhomologous human or non-human sequences; and antibodies in which the FRand/or CDR1 and/or CDR2 regions have been replaced by homologous humanor non-human sequences. In some embodiments, so-called single chainantibodies (e.g., ScFv), (single) domain antibodies, and otherintracellular antibodies may be used in the context of the presentinvention. Domain antibodies, camelid and camelized antibodies andfragments thereof, for example, VHH domains, or nanobodies, such asthose described in patents and published patent applications of AblynxNV and Domantis are also encompassed in the term antibody. Further,chimeric antibodies, e.g., antibodies comprising two antigen-bindingdomains that bind to different antigens, are also suitable for use inthe context of some embodiments of the present invention. In someembodiments, the term antibody may also refer to “antibody mimetics,”which are organic compounds the can specifically bind antigens, but arenot structurally related to antibodies. For example, antibody mimeticsknown as “affibodies,” or “affibody molecules,” are small proteinsengineered to bind e.g., target proteins or peptides with affinitiescomparable to monoclonal antibodies. In some embodiments, an affibodyincludes a protein scaffold based on the Z domain (the immunoglobulin Gbinding domain) of protein A, and in contrast to antibodies, affibodymolecules are composed of alpha helices and lack disulfide bridges.Methods for engineering and producing affibodies are known, and includethose described in Nord et al., “A combinatorial library of an α-helicalbacterial receptor domain.” Prot. Eng. 1995; 8 (6): 601-608; Nord etal., “Binding proteins selected from combinatorial libraries of anα-helical bacterial receptor domain.” Nature Biotechnol. 1997; 15 (8):772-777; Stahl et al., “The use of gene fusions to protein A and proteinG in immunology and biotechnology.” Pathol. Biol. (Paris) 1997; 45 (1):66-76; Rönnmark et al., “Construction and characterization ofaffibody-Fc chimeras produced in Escherichia coli.” J. Immunol. Methods.2002; 261 (1-2): 199-211; Rönnmark et al., “Affibody-beta-galactosidaseimmunoconjugates produced as soluble fusion proteins in the Escherichiacoli cytosol.” J. Immunol. Methods. 2003; 281 (1-2): 149-160; Nord etal., “Recombinant human factor VIII-specific affinity ligands selectedfrom phage-displayed combinatorial libraries of protein A.” Eur. J.Biochem. 2001; 268 (15): 1-10; Engfeldt et al., “Chemical synthesis oftriple-labeled three-helix bundle binding proteins for specificfluorescent detection of unlabeled protein.” Chem. Bio Chem. 2005; 6(6): 1043-1050; Ahlgren et al., “Targeting of HER2-expressing tumorswith a site-specifically 99mTc-labeled recombinant affibody molecule,ZHER2:2395, with C-terminally engineered cysteine.” J. Nucl. Med. 2009;50 (5): 781-789; Orlova et al., “Evaluation of[(111/114m)In]CHX-A”-DTPA-ZHER2:342, an affibody ligand conjugate fortargeting of HER2-expressing malignant tumors.” Q. J. Nucl. Med. Mol.Imaging. 2007; 51 (4): 314-23; Tran et al., “(99m)Tc-maEEE-Z(HER2:342),an Affibody molecule-based tracer for the detection of HER2 expressionin malignant tumors”. Bioconjug. Chem. 2007; 18 (6): 1956-64; Orlova etal., “Tumor imaging using a picomolar affinity HER2 binding affibodymolecule.” Cancer Res. 2006; 66 (8): 4339-48; Holm et al.,“Electrophilic Affibodies Forming Covalent Bonds to Protein Targets.”The Journal of Biological Chemistry 2009; 284 (47): 32906-13; Renberg etal., “Affibody molecules in protein capture microarrays: evaluation ofmultidomain ligands and different detection formats.” J. Proteome Res.2007; 6 (1): 171-179; Lundberg et al., “Site-specifically conjugatedanti-HER2 Affibody molecules as one-step reagents for target expressionanalyses on cells and xenograft samples.” J. Immunol. Methods 2007; 319(1-2): 53-63; Tolmachev et al., “Radionuclide therapy of HER2-positivemicroxenografts using a 177Lu-labeled HER2-specific Affibody molecule.”Cancer Res. 2007; 67 (6): 2773-82; and Gebauer & Skerra, “Engineeredprotein scaffolds as next-generation antibody therapeutics.” CurrentOpinion in Chemical Biology 2009; 13 (3): 245-55; Siontorou C.,“Nanobodies as novel agents for disease diagnosis and therapy.” Int. J.Nanomedicine 2013; 8: 4215-4227; the entire contents of each are herebyincorporated by reference in their entirety.

The term “antigen-binding antibody fragment,” as used herein, refers toa fragment of an antibody that comprises the paratope, or a fragment ofthe antibody that binds to the antigen the antibody binds to, withsimilar specificity and affinity as the intact antibody. Antibodies,e.g., fully human monoclonal antibodies, may be identified using phagedisplay (or other display methods such as yeast display, ribosomedisplay, bacterial display). Display libraries, e.g., phage displaylibraries, are available (and/or can be generated by one of ordinaryskill in the art) that can be screened to identify an antibody thatbinds to an antigen of interest, e.g., using panning. See, e.g., Sidhu,S. (ed.) Phage Display in Biotechnology and Drug Discovery (DrugDiscovery Series; CRC Press; 1^(st) ed., 2005; Aitken, R. (ed.) AntibodyPhage Display: Methods and Protocols (Methods in Molecular Biology)Humana Press; 2nd ed., 2009.

The term “single domain antibody,” as used herein, refers to anantibody, in which the complementarity determining regions (CDRs) arepart of a single domain polypeptide, that is, the complementaritydetermining regions are all on a single polypeptide chain. Examples ofsingle domain antibodies include, but are not limited to, heavy chainantibodies, antibodies naturally devoid of light chains, single domainantibodies derived from conventional 4-chain antibodies, engineeredantibodies, and single domain scaffolds other than those derived fromantibodies. Single domain antibodies may be any of those known in theart, or any future single domain antibodies. Methods for making singledomain antibodies and VHHs are known in the art and would be apparent tothe skilled artisan. For example methods for making single domainantibodies and VHHs are provided in U.S. patent publication No.: US2006/0034845, published on Feb. 16, 2006, the entire contents of whichare incorporated by reference herein. Single domain antibodies may bederived from any species including, but not limited to, mouse, rat,human, camel, llama, goat, rabbit, and bovine. According to one aspectof the invention, a single domain antibody as used herein is a naturallyoccurring single domain antibody known as a heavy chain antibody devoidof light chains. Such single domain antibodies are disclosed inpublished international PCT application, WO 2013/024059, the entirecontents of which are incorporated by reference herein. The variabledomain derived from a heavy chain antibody naturally devoid of lightchain is known as a VHH or nanobody to distinguish it from theconventional VH of four chain immunoglobulins. Such a VHH molecule canbe derived from antibodies raised in Camelidae species, for example,camel, dromedary, llama, vicufia, alpaca, and guanaco. Other speciesbesides Camelidae may produce heavy chain antibodies naturally devoid oflight chain; such VHHs are within the scope of the invention. As usedherein, a “VHH” refers to the variable region of a heavy chain antibody,for example the heavy chain antibody from a camelid. VHHs, useful in thepresent invention and as known to the skilled artisan, may be heavychain variable domains derived from immunoglobulins naturally devoid oflight chains such as those derived from Camelidae as described inpublished international PCT Application, WO 2013/024059, published onFeb. 21, 2013 (and referred to hereinafter as VHH domains ornanobodies). Typically, VHH molecules are about 10× smaller than IgGmolecules. For example, in some embodiments, VHH molecules are between10 kDa and 50 kDa. In some embodiments, VHH molecules are between 10 kDaand 25 kDa. In some embodiments, VHH molecules are between 12 kDa and 16kDa in size. In some embodiments, VHHs lend themselves tosortase-catalyzed enzymatic transformations for a variety of purposes,including the installation of radioisotopes and/or PEG modifications foradministration to a subject and imaging (e.g., PET imaging). They aresingle polypeptides and are stable, resisting extreme pH and temperatureconditions. They have the advantage of specificity, small size, andrapid circulatory clearance (<30 min). In some embodiments, the VHHsprovided herein may be modified (e.g., with a polymer, such as PEG,and/or a detectable label) to yield improved properties for efficientdetection of one or more molecules, proteins, or cells, for example, invivo. Moreover, VHHs are resistant to the action of proteases which isnot the case for conventional antibodies. Furthermore, in vitroexpression of VHHs produces properly folded functional VHHs in highyield. In addition, antibodies generated in Camelids will recognizeepitopes other than those recognized by antibodies generated in vitrothrough the use of antibody libraries or via immunization of mammalsother than Camelids (in published international PCT Application, WO2006/079372, published on Aug. 3, 2006; the entire contents of which areincorporated by reference herein). Since VHHs are known to bind‘unusual’ epitopes such as cavities or grooves (WO 2006/079372), theaffinity of such VHHs may be more suitable for diagnostic purposes.Single domain antibodies useful in the present invention may be producedmy any method known in the art.

The term “binding agent,” as used herein, refers to any molecule thatbinds another molecule with high affinity. In some embodiments, abinding agent binds its binding partner with high specificity. Examplesfor binding agents include, without limitation, antibodies, antibodyfragments, nucleic acid molecules, receptors, ligands, aptamers, andadnectins.

The term “click chemistry” refers to a chemical philosophy introduced byK. Barry Sharpless of The Scripps Research Institute, describingchemistry tailored to generate covalent bonds quickly and reliably byjoining small units comprising reactive groups together (see H. C. Kolb,M. G. Finn and K. B. Sharpless (2001). Click Chemistry: Diverse ChemicalFunction from a Few Good Reactions. Angewandte Chemie InternationalEdition 40 (11): 2004-2021. Click chemistry does not refer to a specificreaction, but to a concept including, but not limited to, reactions thatmimic reactions found in nature. In some embodiments, click chemistryreactions are modular, wide in scope, give high chemical yields,generate inoffensive byproducts, are stereospecific, exhibit a largethermodynamic driving force to favor a reaction with a single reactionproduct, and/or can be carried out under physiological conditions. Insome embodiments, a click chemistry reaction exhibits high atom economy,can be carried out under simple reaction conditions, use readilyavailable starting materials and reagents, uses no toxic solvents oruses a solvent that is benign or easily removed (preferably water),and/or provides simple product isolation by non-chromatographic methods(crystallisation or distillation). In some embodiments, the clickchemistry reaction is a [2+3] dipolar cycloaddition. In certainembodiments, the click chemistry reaction is a Diels-Aldercycloaddition.

The term “click chemistry handle,” as used herein, refers to a reactant,or a reactive group, that can partake in a click chemistry reaction.Exemplary click chemistry handles are demonstrated in U.S. PatentPublication 20130266512, which is incorporated by reference herein. Forexample, a strained alkyne, e.g., a cyclooctyne, is a click chemistryhandle, since it can partake in a strain-promoted cycloaddition (see,e.g., Table 1). In general, click chemistry reactions require at leasttwo molecules comprising click chemistry handles that can react witheach other. Such click chemistry handle pairs that are reactive witheach other are sometimes referred to herein as partner click chemistryhandles. For example, an azide is a partner click chemistry handle to acyclooctyne or any other alkyne. Exemplary click chemistry handlessuitable for use according to some aspects of this invention aredescribed herein, for example, in Tables 1 and 2. In some embodiments,the click chemistry partners are a conjugated diene and an optionallysubstituted alkene, In other embodiments, the click chemistry partnersare an optionally substituted tetrazine and an optionally substitutedtrans-cyclooctene (TCO). In some embodiments, the click chemistrypartners are optionally substituted tetrazine (Tz) and optionallysubstituted trans-cyclooctene (TCO). Tz and TCO react with each other ina reverse-electron demand Diels-Alder cycloaddition reaction (See e.g.,Example 2, FIG. 4; Blackman et al., “The Tetrazine Ligation: FastBioconjugation based on Inverse-electron-demand Diels-Alder Reactivity.”J. Am. Chem. Soc. 2008; 130, 13518-13519). In other embodiments, theclick chemistry partners are an optionally substituted alkyne and anoptionally substituted azide. For example, a difluorinated cyclooctyne,a dibenzocyclooctyne, a biarylazacyclooctynone, or a cyclopropyl-fusedbicyclononyne can be paired with an azide as a click chemistry pair. Inother embodiments, the click chemistry partners are reactive dienes andsuitable tetrazine dienophiles. For example, TCO, norbornene, orbiscyclononene can be paired with a suitable tetrazine dienophile as aclick chemistry pair. In yet other embodiments, tetrazoles can act aslatent sources of nitrile imines, which can pair with unactivatedalkenes in the presence of ultraviolet light to create a click chemistrypair, termed a “photo-click” chemistry pair. The click chemistry pairmay also be a cysteine and a maleimide. For example the cysteine from apeptide (e.g., GGGC (SEQ ID NO: 12)) may be reacted with a maleimidethat is associated with a chelating agent (e.g., NOTA). Other suitableclick chemistry handles are known to those of skill in the art (See,e.g., Table 1; Spicer et al., “Selective chemical protein modification.”Nature Communications. 2014; 5:4740). For two molecules to be conjugatedvia click chemistry, the click chemistry handles of the molecules haveto be reactive with each other, for example, in that the reactive moietyof one of the click chemistry handles can react with the reactive moietyof the second click chemistry handle to form a covalent bond. Suchreactive pairs of click chemistry handles are well known to those ofskill in the art and include, but are not limited to, those described inTable 1.

TABLE 1 Exemplary click chemistry handles and reactions. Exemplary rateconstant (M⁻¹ s⁻¹)

1,3-dipolar cycloaddition 1 x 10^(−3a)

strain-promoted cycloaddition

Diels-Alder reaction

Thiol-ene reaction

Strain-promoted cycloaddition 8 x 10^(−2a)

Strain-promoted cycloaddition     2.3^(a)

Strain-promoted cycloaddition     1^(a)

Strain-promoted cycloaddition     0.1^(a)

Inverse-electron demand Diels-Alder (IEDDA)     9^(a)

Inverse-electron demand Diels-Alder (IEDDA)  17,500^(a)  35,000^(b)

Inverse-electron demand Diels-Alder (IEDDA) >50,000^(a)    880^(b)

1,3-dipolar cycloaddition (“photo-click”)     0.9^(a)

1,3-dipolar cycloaddition (“photo-click”)    58^(a)

Each of R⁴¹, R⁴², and R⁴³ is indpendently hydrogen, optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, optionally substituted heteroaryl, oroptionally substituted heterocyclyl. In certain embodiments, at leastone of R⁴¹, R⁴², and R⁴³ independently comprises a sortase recognitionmotif. In certain embodiments, one of R⁴¹, R⁴², and R⁴³ independentlycomprises a sortase recognition motif. In certain embodiments, two ofR⁴¹, R⁴², and R⁴³ independently comprise a sortase recognition motif. Incertain embodiments, each of R⁴¹, R⁴², and R⁴³ independently comprises asortase recognition motif. In some embodiments, at least one of R⁴¹,R⁴², and R⁴³ is independently R_(R)-LPXT-[X]_(y)- (SEQ ID NO: 22),wherein each occurrence of X independently represents any amino acidresidue; each occurrence of y is an integer between 0 and 10, inclusive;and each occurrence of R_(R) independently represents a protein or anagent (e.g., a protein, peptide, a detectable label, a binding agent, asmall molecule), and, optionally, a linker. Each instance of R₃ isindependently H, substituted or unsubstituted alkyl (e.g., —CH₃), orsubstituted or unsubstituted aryl. ^(a)Exemplary rate constant forsmall-molecule models. ^(b)Exemplary on-protein rate constant.

In some embodiments, click chemistry handles used can react to formcovalent bonds in the absence of a metal catalyst. Such click chemistryhandles are well known to those of skill in the art and include theclick chemistry handles described in Becer, Hoogenboom, and Schubert(Table 2), “Click Chemistry beyond Metal-Catalyzed Cycloaddition,”Angewandte Chemie International Edition (2009) 48: 4900-4908:

TABLE 2 Exemplary click chemistry reactions. Reagent A Reagent BMechanism Notes on reaction^([a]) 0 azide alkyne Cu-catalyzed [3 + 2]azide-alkyne 2 h at 60° C. in H₂O cycloaddition (CuAAC) 1 azidecyclooctyne strain-promoted [3 + 2] azide- 1 h at RT alkynecycloaddition (SPAAC) 2 azide activated [3 + 2] Huisgen cycloaddition 4h at 50° C. alkyne electron- 3 azide deficient [3 + 2] cycloaddittion 12h at RT in H₂O alkyne 4 h at RT in THF with 4 azide aryne [3 + 2]cycloaddition crown ether or 24 h at RT in CH₃CN 5 tetrazine alkeneDiels-Alder retro-[4 + 2] 40 min at 25° C. cycloaddition (100% yield) N₂is the only by-product 6 tetrazole alkene 1,3-dipolar cycloaddition fewmin UV irradiation (photoclick) and then overnight at 4° C. 7dithioester diene hetero-Diels-Alder cycloaddition 10 min at RT 8anthracene maleimide [4 + 2] Diels-Alder reaction 2 days at reflux intoluene 9 thiol alkene radical addition 30 min UV (quantitative (thioclick) conv.) or 24 h UV irradiation (>96%) 10 thiol enone Michaeladdition 24 h at RT in CH₃CN 11 thiol maleimide Michael addition 1 h at40° C. in THF or 16 h at RT in dioxane 12 thiol para-fluoro nucleophilicsubstitution overnight at RT in DMF or 60 min at 40° C. in DMF 13 aminepara-fluoro nucleophilic substitution 20 min MW at 95° C. in NMP assolvent ^([a])RT = room temperature, DMF = N,N-dimethylformamide, NMP =N-methylpyrolidone, THF = tetrahydrofuran, CH₃CN = acetonitrile.

Methods and compositions for using click chemistry in combination withsortagging technologies are known, and include those described by Ploeghet al., international PCT application, PCT/US2012/044584, filed Jun. 28,2012, published as WO 2013/003555 on Jan. 3, 2013; and Ploegh et al.,U.S. patent application U.S. Ser. No. 13/918,278, filed Jun. 14, 2013;the entire contents of each of which are incorporated herein byreference.

The term “conjugated” or “conjugation” refers to an association of twomolecules, for example, two proteins or a protein and an agent, e.g., asmall molecule, with one another in a way that they are linked by adirect or indirect covalent or non-covalent interaction. In certainembodiments, the association is covalent, and the entities are said tobe “conjugated” to one another. In some embodiments, a protein ispost-translationally conjugated to another molecule, for example, asecond protein, a small molecule, a detectable label, a click chemistryhandle, or a binding agent, by forming a covalent bond between theprotein and the other molecule after the protein has been formed, and,in some embodiments, after the protein has been isolated. In someembodiments, two molecules are conjugated via a linker connecting bothmolecules. For example, in some embodiments where two proteins areconjugated to each other to form a protein fusion, the two proteins maybe conjugated via a polypeptide linker, e.g., an amino acid sequenceconnecting the C-terminus of one protein to the N-terminus of the otherprotein. In some embodiments, two proteins are conjugated at theirrespective C-termini, generating a C—C conjugated chimeric protein. Insome embodiments, two proteins are conjugated at their respectiveN-termini, generating an N—N conjugated chimeric protein. In someembodiments, conjugation of a protein to a peptide is achieved bytranspeptidation using a sortase. See, e.g., Ploegh et al.,International PCT Patent Application, PCT/US2010/000274, filed Feb. 1,2010, published as WO/2010/087994 on Aug. 5, 2010, and Ploegh et al.,International Patent Application, PCT/US2011/033303, filed Apr. 20,2011, published as WO/2011/133704 on Oct. 27, 2011, the entire contentsof each of which are incorporated herein by reference, for exemplarysortases, proteins, recognition motifs, reagents, and methods forsortase-mediated transpeptidation. In other embodiments, conjugation ofa protein to a peptide or other moiety may be achieved using otherenzymes known in the art, for example, formylglycine generating enzyme,sialyltransferase, phosphopantetheinyltransferase, transglutaminase,farnesyltransferase, biotin ligase, lipoic acid ligase, or N-myristoyltransferase. Exemplary techniques and approaches for enzymatic labelingof proteins can be found in Rashidian, M., et al. “Enzymatic Labeling ofProteins: Techniques and Approaches”, Bioconjugate Chem., 2013; 24,1277-1294; which is incorporated by reference.

As used herein, a “detectable label” refers to a moiety that has atleast one element, isotope, or functional group incorporated into themoiety which enables detection of the molecule, e.g., a protein orpolypeptide, or other entity, to which the label is attached. Labels canbe directly attached (i.e., via a bond) or can be attached by a tether(such as, for example, an optionally substituted alkylene; an optionallysubstituted alkenylene; an optionally substituted alkynylene; anoptionally substituted heteroalkylene; an optionally substitutedheteroalkenylene; an optionally substituted heteroalkynylene; anoptionally substituted arylene; an optionally substituted heteroarylene;or an optionally substituted acylene, or any combination thereof, whichcan make up a tether). It will be appreciated that the label may beattached to or incorporated into a molecule, for example, a protein,polypeptide, or other entity, at any position.

In general, a label can fall into any one (or more) of five classes: a)a label which contains isotopic moieties, which may be radioactive orheavy isotopes, including, but not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N,¹⁸F, ³¹P, ³²P, ³⁵S, ⁶⁷Ga, ⁷⁶Br, ^(99m)Tc (Tc-99m), ¹¹¹In, ¹²³I, ¹²⁵I,¹³¹I, ¹⁵³Gd, ¹⁶⁹Yb, and ¹⁸⁶Re; b) a label which contains an immunemoiety, which may be antibodies or antigens, which may be bound toenzymes (e.g., such as horseradish peroxidase); c) a label which is acolored, luminescent, phosphorescent, or fluorescent moieties (e.g.,such as the fluorescent label fluoresceinisothiocyanat (FITC); d) alabel which has one or more photo affinity moieties; and e) a labelwhich is a ligand for one or more known binding partners (e.g.,biotin-streptavidin, FK506-FKBP). In certain embodiments, a labelcomprises a radioactive isotope, preferably an isotope which emitsdetectable particles, such as 3 particles. In certain embodiments, thelabel comprises a fluorescent moiety. In certain embodiments, the labelcomprises a fluorophore. In some embodiments, the label is thefluorescent label fluoresceinisothiocyanat (FITC). In certainembodiments, the label comprises a ligand moiety with one or more knownbinding partners. In certain embodiments, the label comprises biotin. Insome embodiments, a label is a fluorescent polypeptide (e.g., GFP or aderivative thereof such as enhanced GFP (EGFP)) or a luciferase (e.g., afirefly, Renilla, or Gaussia luciferase). It will be appreciated that,in certain embodiments, a label may react with a suitable substrate(e.g., a luciferin) to generate a detectable signal. Non-limitingexamples of fluorescent proteins include GFP and derivatives thereof,proteins comprising chromophores that emit light of different colorssuch as red, yellow, and cyan fluorescent proteins, etc. Exemplaryfluorescent proteins include, e.g., Sirius, Azurite, EBFP2, TagBFP,mTurquoise, ECFP, Cerulean, TagCFP, mTFP1, mUkG1, mAG1, AcGFP1, TagGFP2,EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO,mKO2, mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry,mRaspberry, mKate2, mPlum, mNeptune, T-Sapphire, mAmetrine, mKeima. See,e.g., Chalfie, M. and Kain, S R (eds.) Green fluorescent protein:properties, applications, and protocols (Methods of biochemicalanalysis, v. 47). Wiley-Interscience, Hoboken, N.J., 2006, and/orChudakov, D M, et al., Physiol Rev. 90(3):1103-63, 2010 for discussionof GFP and numerous other fluorescent or luminescent proteins. In someembodiments, a label comprises a dark quencher, e.g., a substance thatabsorbs excitation energy from a fluorophore and dissipates the energyas heat.

In some aspects, any of the antibodies (e.g. single domain antibodies)and/or methods of making said antibodies provided herein comprise aradionuclide. In some embodiments, the radionuclide is ²H, ³H, ¹¹C, ¹³C,¹⁴C, ⁶¹Cu, ⁶²Cu, ¹³N, ¹⁵N, ¹⁵O, ¹⁸F, ³¹P, ³²P, ³⁵S, ⁶⁷Ga, ⁶⁸Ga, ⁷⁶Br,^(99m)Tc (Tc-99m), ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵³Gd, ⁸⁹Zr, ⁸⁶Y,¹⁶⁹Yb, ⁸²Rb, or ¹⁸⁶Re. However, in other embodiments, the radionuclideis not ¹⁸F. In other embodiments, the radionuclide is not ⁸⁹Zr.

The term “linker,” as used herein, refers to a chemical group ormolecule covalently linked to a molecule, for example, a protein, and achemical group or moiety, for example, a click chemistry handle. In someembodiments, the linker is positioned between, or flanked by, twogroups, molecules, or moieties and connected to each one via a covalentbond, thus connecting the two. In some embodiments, the linker comprisesone or more atoms. In some embodiments, the linker comprises between 1and 500 atoms. In some embodiments, the linker comprises between 1 and10, 1 and 15, 1 and 20, 1 and 50, 1 and 100, 1 and 150, 1 and 200, 1 and250, 1 and 300, 1 and 350, 1 and 400, and 1 and 450 atoms. In someembodiments, the linker is an amino acid or a plurality of amino acids.In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acids. Insome embodiments, the linker comprises a poly-glycine sequence. In someembodiments, the linker comprises a non-protein structure. In someembodiments, the linker is an organic molecule, group, polymer, orchemical moiety (e.g., polyethylene, polyethylene glycol).

The term “sortase,” as used herein, refers to an enzyme able to carryout a transpeptidation reaction conjugating the C-terminus of a proteinor peptide to the N-terminus of a protein or peptide via transamidation.Sortases are also referred to as transamidases, and typically exhibitboth a protease and a transpeptidation activity. Various sortases fromprokaryotic organisms have been identified. For example, some sortasesfrom Gram-positive bacteria cleave and translocate proteins toproteoglycan moieties in intact cell walls. Among the sortases that havebeen isolated from Staphylococcus aureus, are sortase A (Srt A) andsortase B (Srt B). Thus, in certain embodiments, a transamidase used inaccordance with the present invention is sortase A, e.g., from S.aureus, also referred to herein as SrtA_(aureus). In certainembodiments, a transamidase is a sortase B, e.g., from S. aureus, alsoreferred to herein as SrtB_(aureus).

Sortases have been classified into four classes, designated A, B, C, andD, designated sortase A, sortase B, sortase C, and sortase D,respectively, based on sequence alignment and phylogenetic analysis of61 sortases from Gram-positive bacterial genomes (Dramsi S, Trieu-CuotP, Bierne H, Sorting sortases: a nomenclature proposal for the varioussortases of Gram-positive bacteria. Res Microbiol. 156(3):289-97, 2005;the entire contents of which are incorporated herein by reference).These classes correspond to the following subfamilies, into whichsortases have also been classified by Comfort and Clubb (Comfort D,Clubb R T. “A comparative genome analysis identifies distinct sortingpathways in gram-positive bacteria” Infect Immun., 72(5):2710-22, 2004;the entire contents of which are incorporated herein by reference):Class A (Subfamily 1), Class B (Subfamily 2), Class C (Subfamily 3),Class D (Subfamilies 4 and 5). The aforementioned references disclosenumerous sortases and recognition motifs. See also Pallen, M. J.; Lam,A. C.; Antonio, M.; Dunbar, K. TRENDS in Microbiology, 2001, 9(3),97-101; the entire contents of which are incorporated herein byreference. Those skilled in the art will readily be able to assign asortase to the correct class based on its sequence and/or othercharacteristics such as those described in Drami, et al., supra.

The term “sortase A” is used herein to refer to a class A sortase,usually named SrtA in any particular bacterial species, e.g., SrtA fromS. aureus. Likewise “sortase B” is used herein to refer to a class Bsortase, usually named SrtB in any particular bacterial species, e.g.,SrtB from S. aureus. The invention encompasses embodiments relating to asortase A from any bacterial species or strain. The inventionencompasses embodiments relating to a sortase B from any bacterialspecies or strain. The invention encompasses embodiments relating to aclass C sortase from any bacterial species or strain. The inventionencompasses embodiments relating to a class D sortase from any bacterialspecies or strain.

Amino acid sequences of Srt A and Srt B and the nucleotide sequencesthat encode them are known to those of skill in the art and aredisclosed in a number of references cited herein, the entire contents ofall of which are incorporated herein by reference. The amino acidsequence of a sortase-transamidase from Staphylococcus aureus also hassubstantial homology with sequences of enzymes from other Gram-positivebacteria, and such transamidases can be utilized in the ligationprocesses described herein. For example, for SrtA there is about a 31%sequence identity (and about 44% sequence similarity) with bestalignment over the entire sequenced region of the S. pyogenes openreading frame. There is about a 28% sequence identity with bestalignment over the entire sequenced region of the A. naeslundii openreading frame. It will be appreciated that different bacterial strainsmay exhibit differences in sequence of a particular polypeptide, and thesequences herein are exemplary.

In certain embodiments a transamidase bearing 18% or more sequenceidentity, 20% or more sequence identity, 30% or more sequence identity,40% or more sequence identity, or 50% or more sequence identity with anS. pyogenes, A. naeslundii, S. mutans, E. faecalis or B. subtilis openreading frame encoding a sortase can be screened, and enzymes havingtransamidase activity comparable to Srt A or Srt B from S. aureas can beutilized (e.g., comparable activity sometimes is 10% of Srt A or Srt Bactivity or more).

Thus in some embodiments of the invention the sortase is a sortase A(SrtA). SrtA recognizes the motif (e.g., the sortase recognition motif)LPXTX (wherein each occurrence of X represents independently any aminoacid residue) (SEQ ID NO: 15), with common recognition motifs being,e.g., LPKTG (SEQ ID NO: 23), LPATG (SEQ ID NO: 24), LPNTG (SEQ ID NO:25). In some embodiments LPETG (SEQ ID NO: 11) is used as the sortaserecognition motif. However, motifs falling outside this consensus mayalso be recognized. For example, in some embodiments the motif comprisesan ‘A’ rather than a ‘T’ at position 4, e.g., LPXAG (SEQ ID NO: 17),e.g., LPNAG (SEQ ID NO: 26). In some embodiments the motif comprises an‘A’ rather than a ‘G’ at position 5, e.g., LPXTA (SEQ ID NO: 27), e.g.,LPNTA (SEQ ID NO: 28), e.g., LPETA (SEQ ID NO: 29). In some embodimentsthe motif comprises a ‘G’ rather than ‘P’ at position 2, e.g., LGXTG(SEQ ID NO: 30), e.g., LGATG (SEQ ID NO: 31). In some embodiments themotif comprises an ‘I’ rather than ‘L’ at position 1, e.g., IPXTG (SEQID NO: 32), e.g., IPNTG (SEQ ID NO:33) or IPETG (SEQ ID NO: 34).Additional suitable sortase recognition motifs will be apparent to thoseof skill in the art, and the invention is not limited in this respect.It will be appreciated that the terms “recognition motif” and“recognition sequence”, with respect to sequences recognized by atransamidase or sortase, are used interchangeably.

In some embodiments of the invention the sortase is a sortase B (SrtB),e.g., a sortase B of S. aureus, B. anthracis, or L. monocytogenes.Motifs recognized by sortases (sortase recognition motifs) of the Bclass (SrtB) often fall within the consensus sequences NPXTX (SEQ ID NO:16), e.g., NP[Q/K]-[T/s]-[N/G/s] (SEQ ID NO: 35), such as NPQTN (SEQ IDNO: 36) or NPKTG (SEQ ID NO: 37). For example, sortase B of S. aureus orB. anthracis cleaves the NPQTN (SEQ ID NO:36) or NPKTG (SEQ ID NO:37)motif of IsdC in the respective bacteria (see, e.g., Marraffini, L. andSchneewind, O., Journal of Bacteriology, 189(17), p. 6425-6436, 2007).Other recognition motifs found in putative substrates of class Bsortases are NSKTA (SEQ ID NO: 38), NPQTG (SEQ ID NO: 39), NAKTN (SEQ IDNO:40), and NPQSS (SEQ ID NO: 41). For example, SrtB from L.monocytogenes recognizes certain motifs lacking P at position 2 and/orlacking Q or K at position 3, such as NAKTN (SEQ ID NO: 40) and NPQSS(SEQ ID NO:41) (Mariscotti J F, García-Del Portillo F, Pucciarelli M G.The listeria monocytogenes sortase-B recognizes varied amino acids atposition two of the sorting motif. J Biol Chem. 2009 Jan. 7.)

In some embodiments, the sortase is a sortase C (Srt C). Sortase C mayutilize LPXTX (SEQ ID NO: 15) as a recognition motif, with eachoccurrence of X independently representing any amino acid residue.

In some embodiments, the sortase is a sortase D (Srt D). Sortases inthis class are predicted to recognize motifs with a consensus sequenceNA-[E/A/S/H]-TG (SEQ ID NO: 42) (Comfort D, supra). Sortase D has beenfound, e.g., in Streptomyces spp., Corynebacterium spp., Tropherymawhipplei, Thermobifida fusca, and Bifidobacterium longhum. LPXTA (SEQ IDNO: 43) or LAXTG (SEQ ID NO:44) may serve as a recognition sequence forsortase D, e.g., of subfamilies 4 and 5, respectively subfamily-4 andsubfamily-5 enzymes process the motifs LPXTA (SEQ ID NO: 27) and LAXTG(SEQ ID NO: 44), respectively). For example, B. anthracis Sortase C hasbeen shown to specifically cleave the LPNTA (SEQ ID NO: 28) motif in B.anthracis BasI and BasH (see Marrafini, supra).

See Barnett and Scott for description of a sortase that recognizesQVPTGV (SEQ ID NO: 45) motif (Barnett, T C and Scott, J R, DifferentialRecognition of Surface Proteins in Streptococcus pyogenes by Two SortaseGene Homologs. Journal of Bacteriology, Vol. 184, No. 8, p. 2181-2191,2002; the entire contents of which are incorporated herein byreference). Additional sortases, including, but not limited to, sortasesand sortase variants recognizing additional sortase recognition motifsare also suitable for use in some embodiments of this invention. Forexample, sortases described in Chen I. et al., “A general strategy forthe evolution of bond-forming enzymes using yeast display.” Proc NatlAcad Sci USA. 2011 Jul. 12; 108(28):11399; Dorr, B. M., et al.,“Reprogramming the specificity of sortase enzymes.” Proc. Natl. Acad.Sci. U.S.A. 2014, 111, 13343-13348; the entire contents of each of whichare incorporated herein by reference.

In some embodiments, a variant of a naturally occurring sortase may beused. Such variants may be produced through processes such as directedevolution, site-specific modification, etc. Considerable structuralinformation regarding sortase enzymes, e.g., sortase A enzymes, isavailable, including NMR or crystal structures of SrtA alone or bound toa sortase recognition sequence (see, e.g., Zong Y, et al. J. Biol Chem.2004, 279, 31383-31389). Three dimensional structure information is alsoavailable for other sortases, e.g., S. pyogenes SrtA (Race, P R, et al.,J Biol Chem. 2009, 284(11):6924-33). The active site and substratebinding pocket of S. aureus SrtA have been identified. One of ordinaryskill in the art can generate functional variants by, for example,avoiding deletions or substitutions that would disrupt or substantiallyalter the active site or substrate binding pocket of a sortase. In someembodiments a functional variant of S. aureus SrtA comprises His atposition 120, Cys at position 184, and Arg at position 197, wherein Cysat position 184 is located within a TLXTC (SEQ ID NO: 46) motif.Functional variants of other SrtA proteins may have His, Cys, Arg, andTLXTC (SEQ ID NO: 46) motifs at positions that correspond to thepositions of these residues in S. aureus SrtA. In some embodiments, asortase variant comprises a sequence at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a wild type sortase Asequence or catalytic domain thereof, e.g., at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids60-206 of SEQ ID NO: 47 or SEQ ID NO: 48, or at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids26-206 of SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, a sortasevariant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15amino acid substitutions relative to amino acids 60-206 of SEQ ID NO: 47or relative to amino acids 26-206 of SEQ ID NO: 47 or SEQ ID NO: 48.

In some embodiments, a transamidase having higher transamidase activitythan a naturally occurring sortase may be used. In some embodiments theactivity of the transamidase is at least about 10, 15, 20, 40, 60, 80,100, 120, 140, 160, 180, or 200 times as high as that of S. aureussortase A. In some embodiments the activity is between about 10 and 50times as high as that of S. aureus sortase A, e.g., between about 10 and20 times as high, between about 20 and 30 times as high, between about30 and 50 times as high. In some embodiments the activity is betweenabout 50 and about 150 times as high as that of S. aureus sortase A,e.g., between about 50 and 75 times as high, between about 75 and 100times as high, between about 100-125 times as high, or between about 125and 150 times as high. For example, variants of S. aureus sortase A withup to a 140-fold increase in LPETG-coupling (SEQ ID NO: 11) activitycompared with the starting wild-type enzyme have been identified (Chen,I., et al., PNAS 108(28): 11399-11404, 2011). In some embodiments such asortase variant is used in a composition or method of the invention. Insome embodiments a sortase variant comprises any one or more of thefollowing substitutions relative to a wild type S. aureus SrtA: P94S orP94R, D160N, D165A, K190E, and K196T mutations.

One of ordinary skill in the art will appreciate that the foregoingdescriptions of substitutions utilize standard notation of the formX₁NX₂, in which X₁ and X₂, represent amino acids and N represents anamino acid position, X₁ represents an amino acid present in a firstsequence (e.g., a wild type S. aureus SrtA sequence), and X₂ representsan amino acid that is substituted for X₁ at position N, resulting in asecond sequence that has X₂ at position N instead of X₁. It should beunderstood that the present disclosure is not intended to be limited inany way by the identity of the original amino acid residue X₁ that ispresent at a particular position N in a wild type SrtA sequence used togenerate a SrtA variant and is replaced by X₂ in the variant. Anysubstitution which results in the specified amino acid residue at aposition specified herein is contemplated by the disclosure. Thus asubstitution may be defined by the position and the identity of X₂,whereas X₁ may vary depending, e.g., on the particular bacterial speciesor strain from which a particular SrtA originates. Thus in someembodiments, a sortase A variant comprises any one or more of thefollowing: an S residue at position 94 (S94) or an R residue at position94 (R94), an N residue at position 160 (N160), an A residue at position165 (A165), an E residue at position 190 (E190), a T residue at position196 (T196) (numbered according to the numbering of a wild type SrtA,e.g., SEQ ID NO: 47). For example, in some embodiments a sortase Avariant comprises two, three, four, or five of the afore-mentionedmutations relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 47).In some embodiments a sortase A variant comprises an S residue atposition 94 (S94) or an R residue at position 94 (R94), and also an Nresidue at position 160 (N160), an A residue at position 165 (A165), anda T residue at position 196 (T196). For example, in some embodiments asortase A variant comprises P94S or P94R, and also D160N, D165A, andK196T. In some embodiments a sortase A variant comprises an S residue atposition 94 (S94) or an R residue at position 94 (R94) and also an Nresidue at position 160 (N160), A residue at position 165 (A165), a Eresidue at position 190, and a T residue at position 196. For example,in some embodiments a sortase A variant comprises P94S or P94R, and alsoD160N, D165A, K190E, and K196T. In some embodiments a sortase A variantcomprises an R residue at position 94 (R94), an N residue at position160 (N160), a A residue at position 165 (A165), E residue at position190, and a T residue at position 196. In some embodiments a sortasecomprises P94R, D160N, D165A, K190E, and K196T.

It is to be further understood that the disclosure contemplates variantsof any wild-type sortase A. Those skilled in the art will appreciatethat wild-type sequences of sortase A may vary, e.g., SrtA from variousspecies may have gaps, insertions, and/or may vary in length relative tothe amino acid sequence of exemplary wild-type S. aureus SrtA. Thoseskilled in the art will appreciate that the positions described hereinin regard to substitutions or other alterations pertain to the sequenceof exemplary wild type S. aureus SrtA, unless otherwise indicated, andthat such positions may be adjusted when making correspondingsubstitutions in different bacterial SrtA sequences in order to accountfor such gaps, insertions, and/or length differences. For example, asnoted above, certain sortase variants comprise a substitution at aminoacid position 94 (e.g., the amino acid is changed to an S residue).However, the amino acid at position 94 in S. aureus SrtA may correspondto an amino acid at a different position (e.g., position Z) in SrtA froma second bacterial species when the sequences are aligned. Whengenerating a variant of the SrtA of the second bacterial speciescomprising a substitution at “position 94” (based on the wild type S.aureus SrtA sequence numbering), it is the amino acid at position Z ofthe SrtA from the second bacterial species that should be changed (e.g.,to S) rather than the amino acid at position 94. Those skilled in theart will understand how to align any original wild-type sortase Asequence to be used for generating a SrtA variant with an exemplarywild-type S. aureus sortase A sequence for purposes of determining thepositions in the original wild-type sortase A sequence that correspondto the exemplary wild-type S. aureus sortase A sequence when taking intoaccount gaps and/or insertions in the alignment of the two sequences.

In some embodiments, amino acids at position 94, 160, 165, 190, and/or196 are altered in a variant as compared with the amino acids present atthose positions in a wild type S. aureus SrtA, and the other amino acidsof the variant are identical to those present at the correspondingpositions in a wild type SrtA, e.g., a wild type S. aureus SrtA. In someembodiments, one or more of the other amino acids of a variant, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 of the other amino acids differ from thosepresent at corresponding position(s) in a wild type SrtA, e.g., a wildtype S. aureus SrtA. In some embodiments a variant may have any of theproperties or degrees of sequence identity specified in the definitionof “variants” above.

An exemplary wild type S. aureus SrtA sequence (Gene ID: 1125243, NCBIRefSeq Acc. No. NP_375640.1) is shown below, with the afore-mentionedpositions underlined:

(SEQ ID NO: 47) MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYDKNVKEQASKDNKQQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVEVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIF VATEVK.

One of ordinary skill in the art will appreciate that differentsubspecies, strains, and isolates may differ in sequence at positionsthat do not significantly affect activity. For example, anotherexemplary wild type S. aureus SrtA sequence (Gene ID: 3238307, NCBIRefSeq Acc. No. YP_187332.1; GenBank Acc. No. AAD48437) has a K residueat position 57 and a G residue at position 167, as shown below in SEQ IDNO: 48:

(SEQ ID NO: 48) MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYDKNVKEQASKDKKQQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIF VATEVK

Either or both of these amino acids (i.e., K57 and/or G167) may bepresent in or introduced into any SrtA sequence, e.g., any S. aureusSrtA sequence, whether naturally occurring or generated by man.Furthermore, as described herein, any sortase sequence may furthercomprise a tag (e.g., 6×His), a spacer, or both. For example, the N- orC-terminus may be extended to encompass a tag, optionally separated fromthe rest of the sequence by a spacer,

In some embodiments a sortase variant comprising the following sequencemay be used, in which amino acid substitutions relative to a wild typeS. aureus SrtA of SEQ ID NO: 47 or SEQ ID NO: 48 are shown in underlinedbold letters:

(SEQ ID NO: 49) MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPAT R EQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSI R N VKPT AVEVLDEQKGKDKQLTLITCDDYNE E TGVWE T RKIFVATEVK.

As will be appreciated, amino acids 2-148 of the above sequencecorrespond to amino acids 60-206 of the full length S. aureus SrtAsequence (the catalytic domain). For example, the “R” residue atposition 36 of SEQ ID NO: 49 corresponds to the “P” residue at position94 in SEQ ID NO: 47 or 48. It is also contemplated in some embodimentsto use sortase variants that have other substitutions at one or more ofpositions 94, 160, 165, 190, and 196 (numbered according to thenumbering of SEQ ID NO: 47 or 48), e.g., wherein such substitutionsutilize an amino acid that would be a conservative substitution at therelevant position as compared with the sequence of SEQ ID NO: 49.

The use of sortases found in any gram-positive organism, such as thosementioned herein and/or in the references (including databases) citedherein is contemplated in the context of some embodiments of thisinvention. Also contemplated is the use of sortases found in gramnegative bacteria, e.g., Colwellia psychrerythraea, Microbulbiferdegradans, Bradyrhizobium japonicum, Shewanella oneidensis, andShewanella putrefaciens. Such sortases recognize sequence motifs outsidethe LPXTX (SEQ ID NO: 15) consensus, for example, LP[Q/K]T[A/S]T (SEQ IDNO: 50). In keeping with the variation tolerated at position 3 insortases from gram-positive organisms, a sequence motif LPXT[A/S] (SEQID NO: 51), e.g., LPXTA (SEQ ID NO:27) or LPSTS (SEQ ID NO: 52) may beused.

Those of skill in the art will appreciate that any sortase recognitionmotif known in the art can be used in some embodiments of thisinvention, and that the invention is not limited in this respect. Forexample, in some embodiments the sortase recognition motif is selectedfrom: LPKTG (SEQ ID NO: 23), LPITG (SEQ ID NO: 53), LPDTA (SEQ ID NO:54), SPKTG (SEQ ID NO:55), LAETG (SEQ ID NO: 56), LAATG (SEQ ID NO: 57),LAHTG (SEQ ID NO: 58), LASTG (SEQ ID NO: 59), LPLTG (SEQ ID NO: 60),LSRTG (SEQ ID NO: 61), LPETG (SEQ ID NO:11), VPDTG (SEQ ID NO: 62),IPQTG (SEQ ID NO: 63), YPRRG (SEQ ID NO: 64), LPMTG (SEQ ID NO:65),LAFTG (SEQ ID NO: 66), LPQTS (SEQ ID NO: 67), it being understood thatin various embodiments of the invention the fifth residue may bereplaced with any other amino acid residue. For example, the sequenceused may be LPXT (SEQ ID NO: 22), LAXT (SEQ ID NO: 68), LPXA (SEQ ID NO:69), LGXT (SEQ ID NO: 70), IPXT (SEQ ID NO: 71), NPXT (SEQ ID NO: 72),NPQS (SEQ ID NO: 73), LPST (SEQ ID NO: 74), NSKT (SEQ ID NO: 75), NPQT(SEQ ID NO: 76), NAKT (SEQ ID NO: 77), LPIT (SEQ ID NO: 78), LAET (SEQID NO: 79), or NPQS (SEQ ID NO: 80). The invention encompassesembodiments in which ‘X’ in any sortase recognition motif disclosedherein or known in the art is amino acid, for example, any naturallyoccurring or any non-naturally occurring amino acid. In someembodiments, X is selected from the 20 standard amino acids found mostcommonly in proteins found in living organisms. In some embodiments,e.g., where the recognition motif is LPXTG (SEQ ID NO: 81) or LPXT (SEQID NO: 22), X is D, E, A, N, Q, K, or R. In some embodiments, X in aparticular recognition motif is selected from those amino acids thatoccur naturally at position 3 in a naturally occurring sortasesubstrate. For example, in some embodiments X is selected from K, E, N,Q, A in an LPXTG (SEQ ID NO: 81) or LPXT (SEQ ID NO: 22) motif where thesortase is a sortase A. In some embodiments X is selected from K, S, E,L, A, N in an LPXTG (SEQ ID NO: 81) or LPXT (SEQ ID NO: 22) motif and aclass C sortase is used.

In some embodiments, a sortase recognition sequence further comprisesone or more additional amino acids, e.g., at the N- or C-terminus. Forexample, one or more amino acids (e.g., up to five amino acids) havingthe identity of amino acids found immediately N-terminal to, orC-terminal to, a five amino acid recognition sequence in a naturallyoccurring sortase substrate may be incorporated. Such additional aminoacids may provide context that improves the recognition of therecognition motif.

The term “sortase recognition motif,” as used herein, refers to anymolecule that is recognized by a sortase, for example, any molecule thatcan partake in a sortase-mediated transpeptidation reaction. In someembodiments, “sortase recognition motif” and “sortase recognitionsequence” are used interchangeably. A typical sortase-mediatedtranspeptidation reaction involves a substrate comprising a C-terminalsortase recognition motif, e.g., an LPXTX (SEQ ID NO: 15) motif, and asecond substrate, referred to herein as a “sortase substrate.” A sortasesubstrate is a chemical moiety that can partake in a sortase-mediatedtranspeptidation reaction with a sortase recognition motif. In someembodiments, the sortase substrate is a polyglycine or polyalanine. Insome embodiments, the sortase substrate comprises an alkylamine group.In some embodiments, the sortase substrate is N-terminal. In someembodiments, the sortase substrate is C-terminal In some embodiments, asortase substrate, or substrate recognition motif though described asbeing “C-terminal” or N-terminal,” is not required to be at theimmediate C- or N-terminus. For example, in some embodiments, otheramino acids, for example a tag (e.g., a 6×His-tag), are found at theimmediate C-terminus of a protein comprising a C-terminal sortaserecognition motif, and the C-terminal sortase recognition motif isadjacent (e.g., within 5, 10, 15 or 20 amino acids) thereto. A sortaserecognition motif may be a peptide or a protein, for example, a peptidecomprising a sortase recognition motif such as an LPXTX (SEQ ID NO: 15).In some embodiments, a sortase substrate is a polyglycine a polyalanine,or an alkylamine group wherein the peptide is conjugated to an agent,e.g., a radiolabeled compound or small molecule. Accordingly, bothproteins and non-protein molecules can be sortase substrates. Someexamples of sortase substrates are described in more detail elsewhereherein and additional suitable sortase substrates will be apparent tothe skilled artisan. The invention is not limited in this respect.

The term “sortagging,” as used herein, refers to the process of adding atag or agent, e.g., a moiety or molecule, for example, a radiolabeledcompound or small molecule, onto a target molecule, for example, atarget protein for use in PET applications via a sortase-mediatedtranspeptidation reaction. Examples of additional suitable tags include,but are not limited to, amino acids, nucleic acids, polynucleotides,sugars, carbohydrates, polymers, lipids, fatty acids, and smallmolecules. Other suitable tags will be apparent to those of skill in theart and the invention is not limited in this aspect. In someembodiments, a tag comprises a sequence useful for purifying,expressing, solubilizing, and/or detecting a polypeptide. In someembodiments, a tag can serve multiple functions. In some embodiments, atag comprises an HA, TAP, Myc, 6×His, Flag, streptavidin, biotin, or GSTtag, to name a few examples. In some embodiments, a tag is cleavable, sothat it can be removed, e.g., by a protease. In some embodiments, thisis achieved by including a protease cleavage site in the tag, e.g.,adjacent or linked to a functional portion of the tag. Exemplaryproteases include, e.g., thrombin, TEV protease, Factor Xa, PreScissionprotease, etc. In some embodiments, a “self-cleaving” tag is used. See,e.g., Wood et al., International PCT Application PCT/US2005/05763, filedon Feb. 24, 2005, and published as WO/2005/086654 on Sep. 22, 2005.

The term “subject” includes, but is not limited to, vertebrates, morespecifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep,goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a birdor a reptile or an amphibian. In some embodiments, the subject is ahuman subject. As used herein, “patient” refers to a subject afflictedwith a disease or disorder. The term “patient” includes human andveterinary subjects of any sex or stage of development.

The term “target protein,” as used herein in the context ofsortase-mediated modification of proteins, refers to a protein that ismodified by the conjugation of an agent, for example a radioactive agentthat renders the protein suitable for diagnostic and/or therapeuticapplications, such as PET imaging. The term “target protein” may referto a wild type or naturally occurring form of the respective protein, orto an engineered form, for example, to a recombinant protein variantcomprising a sortase recognition motif not contained in a wild-type formof the protein. The term “modifying a target protein,” as used herein inthe context of sortase-mediated protein modification, refers to aprocess of altering a target protein comprising a sortase recognitionmotif via a sortase-mediated transpeptidation reaction. Typically, themodification leads to the target protein being conjugated to an agent,for example, a peptide, protein, detectable label, or small molecule. Incertain embodiments, the modification provides radiolabeled proteins.

Antibodies and Single Domain Antibodies

Some aspects of the disclosure provide antibodies (e.g., single domainantibodies) comprising a hydrophilic polymer and a detectable label.Other aspects of the disclosure provide methods for conjugatinghydrophilic polymers and/or detectable labels to antibodies (e.g.,single domain antibodies). In some embodiments, such antibodies areuseful for imaging, for example a tumor, a tissue, or an organ in asubject. It should be appreciated that any of the antibodies providedherein may bind to a particular antigen. In some embodiments, any of theantibodies provided herein may be produced, for example, by immunizing asubject (e.g., research animal) with an antigen, or using other methods,such as phage display. In some embodiments, an antibody is said to bindto an antigen if it is capable of binding to said antigen with anaffinity better than 10⁻⁶M. In some embodiments, an antibody is said tobind to an antigen if it is capable of binding to said antigen with anaffinity better than 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, or 10⁻¹¹M.

Any of the antibodies (e.g., single domain antibodies) provided hereinmay be humanized. Methods of humanizing antibodies are known in the artand would be apparent to the skilled artisan. Technology to humanizecamelid VHHs has been developed (see e.g., Vincke et al., J. Biol. Chem.2009, 284, 3273-3284; which is incorporated herein by reference), andseveral VHHs have been used already in a number of phase I and phase IIclinical trials for therapeutic applications (see, e.g., De Meyer etal., Trends Biotechnol. 2014, 32, 263-270; which is incorporated hereinby reference). Accordingly, in some embodiments, methods for using VHHs(e.g., any of the VHHs provided herein) are provided. In someembodiments, the methods include detecting an epitope in a subject. Insome embodiments, the methods include detecting a lymphocyte, e.g., in atumor of a subject. In some embodiments, the methods include determiningwhether a subject will respond to a therapeutic agent.

In some embodiments, any of the antibodies provided herein bind to anyimmune cell. Some examples of immune cells include T-cells, B-cells,plasma cells, macrophages, dendritic cells, neutrophils, eosinophils, ormast cells. In some embodiments, the antibody binds to a marker ofinflammation. For example, in some embodiments, the antibody useful indiagnostic applications involving PET. Use of antibodies for PET basedapplications is referred to as immunoPET (See, e.g., Knowles et al.,“Advances in immuno-positron emission tomography: antibodies formolecular imaging in oncology.” J Clin Oncol. 2012; 30:3884-3892; theentire contents of which are hereby incorporated by reference). Suchantibodies include monoclonal antibodies known to target or bindcancerous cells or tissues in a subject's body. For example, anon-limiting list of antibodies approved by the U.S. Food and DrugAdministration (FDA) and the European Medicines Agency is provided inTable 3 of Salsano and Treglia, “PET imaging using radiolabeledantibodies: future direction in tumor diagnosis and correlateapplications.” Research and Reports in Nuclear Medicine. 2013: 3; 9-17,the entire contents of which are hereby incorporated by reference. Thetable is reproduced below.

TABLE 3 List of monoclonal antibodies approved by the US Food and DrugAdministration and the European Medicines Agency in cancer therapy.Brand Target: Approval year Antibody name Type antibody type ApplicationCompany EU USA Rituximab Rituxan, Chimeric IgG1 CD20 Non-HodgkinGenentech 1998 1997 MabThera lymphoma Trastuzumab Herceptin HumanizedIgG1 HER2 Beast cancer Genentech/Roche 2000 1998 Gemtuzumab Mylotarg*Humanized IgG4, CD33 Acute myeloid Wyeth/Pfizer NA 2000 ozogamicinimmunotoxin leukemia Alemtuzumab MabCampath, Humanized IgG1 CD52 Chronicmyeloid Genzyme 2001 2001 Campath-IH leukemia Ibritumomab Zevalin MurineIgG1 CD20 Non-Hodgkin Biogen Idec 2004 2002 tiuxecan lymphomaTositumomab Bexxar Murine IgG2a CD20 Non-Hodgkin Corixa/GSK NA 2003lymphoma Cecuximab Erbitux Chimeric IgG1 EGFR Colorectal cancer,Imclone/Lilly 2004 2004 head and neck cancer Bevacizumab AvastinHumanized IgG1 VEGF Colorectal cancer, Genentech/Roche 2005 2004non-small cell lung cancer Panitumumab Vectibix Human IgG2 EGFRColorectal cancer Amgen 2007 2006 Ofatumumab Arzerra Human IgG1 CD20Chronic lymphocytic Genmab 2010 2009 leukemia Denosumab Prolia HumanIgG2 RANK ligand Bone metastases, Amgen 2010 2010 giant cell tumor ofbone Ipilimumab Yervoy Human IgG1 CTLA-4 Melanoma BMS 2011 2011Brentuximab Adcetris Chimeric IgG1, CD30 Anaplastic large SeattleGenetics 2012 2011 vedotin drug-conjugate cell lymphoma, Hodgkinlymphoma Pertuzumab Perjeta Humanized IgG1 HER2 Breast cancerGenentech/Roche 2013 2012 Ado-trastuzumab Kadeyla Humanized IgG1, HER2Breast cancer Genentech/Roche in review 2013 emtansine drug-conjugateNote: *withdrawn in 2010. Abbreviations: CTLA 4, cytotoxic T-lymphocyteantigen 4; EGFR, epidermal growth factor receptor; HER, human epidermalreceptor; NA, not approved; VEGF, vascular endothelial growth factor.

Any of the antibodies, or fragments thereof, disclosed in Table 3 ofSalsano and Treglia can be labeled according to the methods providedherein. Other antibodies amenable to labeling as described hereininclude, but are not limited to, those described in Wright and Lapi,“Designing the magic bullet? The advancement of immuno-PET into clinicaluse.” J. Nucl. Med. 2013 August; 54(8):1171-4; the entire contents ofwhich are hereby incorporated by reference. These antibodies (see below)were successfully labeled with isotopes and were used in PET baseddiagnostic and/or therapeutic applications. However, the antibodies werelabeled via chemical means that are not always amenable to quicklygenerating labeled antibodies with isotopes having a short half-life.Thus, such antibodies can be quickly and efficiently labeled with anydesired isotope according to the methods, compositions, reagents, andkits provided herein. Antibodies disclosed by Wright and Lapi, include:

Humanized A33 (huA33), which recognizes A33 antigen, which is known tobe expressed in greater than 95% of human colon adenocarcinomas. In astudy utilizing radiolabeled huA33 (Carrasquillo et al., “¹²⁴I-huA33antibody PET of colorectal cancer.” J. Nucl. Med. 2011; 52:1173-1180;the entire contents of which are hereby incorporated by reference), 25patients with primary or metastatic colorectal cancer (CRC) wereadministered 44.4-396 MBq (median, 343 MBq) of ¹²⁴I-huA33 with a totalof 10 mg of huA33. No adverse side effects were observed during thetreatment that could be attributed to the huA33. The antibody could beadministered via intravenous administration or hepatic arterial infusion(HAI), with HAI giving no detectable advantage over intravenousinjection. Eleven patients had 12 primary tumors, 10 of which weredetected via immuno-PET. Ten patients had liver metastases, all of whichwere detected by ¹²⁴I-huA33. Four of 7 patients with nodal metastasesdisplayed uptake of the ¹²⁴I-huA33, and 2 of 5 patients had lung lesionsthat were visualized by immuno-PET.

Radretumab (L19SIP), which targets an epitope contained in theextra-domain B of fibronectin, was labeled with ¹²⁴I and used toestablish provisional doses of ¹³¹I-labeled radretumab in 6 patientswith brain metastasis (Poli et al., “Radretumab radioimmunotherapy inpatients with brain metastasis: a ¹²⁴I-L19SIP dosimetric PET study.Cancer Immunol Res. 2013:OF1-OF10; the entire contents of which arehereby incorporated by reference).

Girentuximab (cG250), a chimeric antibody that binds carbonic anhydraseIX (CAIX), expressed in >95% of clear cell renal carcinoma (ccRCC), waslabeled with ¹²⁴I and used to detect such cancers (Divgi et al.,“Positron emission tomography/computed tomography identification ofclear cell renal cell carcinoma: results from the REDECT Trial.” J.Clin. Oncol. 2013; 31:187-194; the entire contents of which are herebyincorporated by reference).

Panitumumab, a fully humanized antibody that binds epidermal growthfactor receptor (EGFR), was successfully labeled with ⁸⁹Zr and used toimage colorectal tumor xenografts (Nayak et al., “PET and MR imaging ofmetastatic peritoneal and pulmonary colorectal cancer in mice with humanepidermal growth factor receptor 1-targeted ⁸⁹Zr-Labeled panitumumab.”J. Nucl. Med. 2012; 53:113-120; Chang et al., “Development andcharacterization of ⁸⁹Zr-labeled panitumumab for immuno-positronemission tomographic imaging of the epidermal growth factor receptor.”Mol. Imaging. 2013; 12:17-27; the entire contents of each are herebyincorporated by reference).

U36, a chimeric antibody that recognizes the v6 region of CD44, waslabeled with ⁸⁹Zr to image head and neck squamous cell carcinoma(Birjesson et al. “Radiation dosimetry of ⁸⁹Zr-labeled chimericmonoclonal antibody U36 as used for immuno-PET in head and neck cancerpatients.” J. Nucl. Med. 2009; 50:1828-1836; the entire contents ofwhich are hereby incorporated by reference).

Trastuzumab, cetuximab, and bevacizumab (see Table 4 above), were alsosuccessfully labeled with ⁸⁹Zr and used in PET applications (Dijkers etal., “Biodistribution of ⁸⁹Zr-trastuzumab and PET imaging ofHER2-positive lesions in patients with metastatic breast cancer.” Clin.Pharmacol. Ther. 2010; 87:586-592;www.cancer.gov/clinicaltrials/search/results?protocolsearchid511815785.Accessed Jul. 15, 2013; the entire contents of each are herebyincorporated by reference).

In some embodiments, any of the antibodies provided herein binds to atumor antigen. In general, a tumor antigen can be any antigenicsubstance produced by a tumor cell (e.g., tumorigenic cells, or in someembodiments tumor stromal cells, e.g., tumor-associated cells such ascancer-associated fibroblasts). In many embodiments, a tumor antigen isa molecule (or portion thereof) that is differentially expressed bytumor cells as compared with non-tumor cells. In other embodiments, atumor antigen is expressed on the surface of the cell. Tumor antigensmay include, e.g., proteins that are normally produced in very smallquantities and are expressed in larger quantities by tumor cells,proteins that are normally produced only in certain stages ofdevelopment, proteins whose structure (e.g., sequence orpost-translational modification(s)) is modified due to a mutation intumor cells, or normal proteins that are (under normal conditions)sequestered from the immune system. Tumor antigens may be useful in,e.g., identifying or detecting tumor cells (e.g., for purposes ofdiagnosis and/or for purposes of monitoring subjects who have receivedtreatment for a tumor, e.g., to test for recurrence) and/or for purposesof targeting various agents (e.g., therapeutic agents) to tumor cells.For example, in some embodiments, a radiolabeled antibody is providedcomprising an antibody or antibody fragment that binds a tumor antigen,thereby allowing detection of the tumor in vivo, e.g., using PET. Insome embodiments, a tumor antigen is an expression product of a mutatedgene, e.g., an oncogene or mutated tumor suppressor gene, anoverexpressed or aberrantly expressed cellular protein, an antigenencoded by an oncogenic virus (e.g., HBV; HCV; herpesvirus familymembers such as EBV, KSV; papilloma virus, etc.), or an oncofetalantigen. Oncofetal antigens are normally produced in the early stages ofembryonic development and largely or completely disappear by the timethe immune system is fully developed. Examples are alphafetoprotein(AFP, found, e.g., in germ cell tumors and hepatocellular carcinoma) andcarcinoembryonic antigen (CEA, found, e.g., in bowel cancers andoccasionally in lung and breast cancers). Tyrosinase is an example of aprotein normally produced in very low quantities but whose production isgreatly increased in certain tumor cells (e.g., melanoma cells). Otherexemplary tumor antigens include, e.g., CA-125 (found, e.g., in ovariancancer); MUC-1 (found, e.g., in breast cancer); epithelial tumor antigen(found, e.g., in breast cancer); melanoma-associated antigen (MAGE;found, e.g., in malignant melanoma); and prostatic acid phosphatase(PAP, found in prostate cancer). In some embodiments, a tumor antigen isat least in part exposed at the cell surface of tumor cells. In someembodiments, a tumor antigen comprises an abnormally modifiedpolypeptide or lipid, e.g., an aberrantly modified cell surfaceglycolipid or glycoprotein. It will be appreciated that a tumor antigenmay be expressed by a subset of tumors of a particular type and/or by asubset of cells in a tumor. Additional exemplary tumor antigens areknown in the art and are within the scope of this disclosure. Forexample exemplary tumor antigens are described in WO 2014/183066 and US20160122707, the entire contents of each are incorporated by referenceherein.

Other exemplary therapeutic/diagnostic antibodies, or fragments thereof,that are useful in the production of radiolabeled antibodies or proteinsaccording to the methods provided herein include, but are not limitedto, the following antibodies, or fragments thereof (the target of theantibody is listed in parentheses together with exemplary non-limitingtherapeutic indications):

Abciximab (glycoprotein IIb/IIIa; cardiovascular disease), Adalimumab(TNF-α, various auto-immune disorders, e.g., rheumatoid arthritis),Alemtuzumab (CD52; chronic lymphocytic leukemia), Basiliximab (IL-2Rαreceptor (CD25); transplant rejection), Bevacizumab (vascularendothelial growth factor A; various cancers, e.g., colorectal cancer,non-small cell lung cancer, glioblastoma, kidney cancer; wet age-relatedmacular degeneration), Catumaxomab (CD3 and EpCAM, malignant ascites),Cetuximab (EGF receptor, various cancers, e.g., colorectal cancer, headand neck cancer), Certolizumab (e.g., Certolizumab pegol) (TNF alpha;Crohn's disease, rheumatoid arthritis), Daclizumab (IL-2Rα receptor(CD25); transplant rejection), Eculizumab (complement protein C5;paroxysmal nocturnal hemoglobinuria), Efalizumab (CD11a; psoriasis),Gemtuzumab (CD33; acute myelogenous leukemia (e.g., conjugated tocalicheamicin)), Ibritumomab tiuxetan (CD20; Non-Hodgkin lymphoma (e.g.,labeled with yttrium-90 or indium-111)), Infliximab (TNF alpha; variousautoimmune disorders, e.g., rheumatoid arthritis) Muromonab-CD3 (T CellCD3 receptor; transplant rejection), Natalizumab (alpha-4 (α4) integrin;multiple sclerosis, Crohn's disease), Omalizumab (IgE; allergy-relatedasthma), Palivizumab (epitope of RSV F protein; Respiratory SyncytialVirus infection), Panitumumab (EGF receptor; cancer, e.g., colorectalcancer), Ranibizumab (vascular endothelial growth factor A; wetage-related macular degeneration) Rituximab (CD20; non-Hodgkinlymphoma), Tositumomab (CD20; non-Hodgkin lymphoma), Trastuzumab (ErbB2;breast cancer), and any antigen-binding fragments thereof.

In some embodiments, any of the antibodies, or fragments thereof, (e.g.,radiolabeled and/or PEGylated), described herein, may be used to imagean immune response. The in vivo imaging of the inflammatory response,e.g., by labeling sites of inflammation using the methods andcompositions provided herein, allows for non-invasive diagnosis,monitoring, and treatment of inflammatory disorders, as describedherein. Other exemplary inflammatory markers to which radiolabeledproteins of the instant disclosure may bind include, but are not limitedto, cytokines, tumor necrosis factor (TNF)-α, IL-6, IL-1 beta, IL-8,IL-10, IL-12, IL-16, IL-18, monocyte chemoattractant protein-1 (MCP-1),GRO-α (Growth Related Oncogene-α), matrix metalloproteinase-8 (MMP-8),CSFs (colony-stimulating factors), epithelial cell-derivedneutrophil-activating peptide-78 (ENA-78), regulated on activationnormal T cell expressed and secreted (RANTES) CCL5, CXCL6 (granulocytechemotactic protein-2), CXCL9 MIG, CXCL10; IP-10, CXCL11, CXCL13(BCA-1), Exodus-1 (CCL20), MIF (macrophage migration inhibitory factor):MIP-1alpha (CCL3), MIP-1beta (CCL4), CD11b, CD11c, CD13, CD15, CD66,CD14, CD64, CD66b, CD18, CD16, CD62L, CD67, HLA-DR, sHLA-G,Dihydroepiandrotendione (DHEA)-S, Cortisol CRF (corticotrophin-releasingfactor), CRF-binding protein, alpha-defensin, beta-defensin, neutrophildefensins (HNP 1-3), bactericidal/permeability-increasing protein (BPI),calprotectin (MRP8/14), surfactant protein-A, surfactant protein-D,serum amyloid P component, serum amyloid A, complement factors,mannan-binding lectin, fibrinogen, prothrombin, factor VIII, vonWillebrand factor, plasminogen, mannan-bindinglectin, c-reactiveprotein, Pentraxin 3, scavenger receptors, C-type lectins, Toll-likereceptor (TLR)-4, TLR-2, TLR-3, TLR-6, intracellular pattern recognitionreceptors (Nod1, Nod2, RIG-1, MDA-5), RAGE (receptor for advancedglycation endproduct), alpha 2-macroglobulin, ferritin, hepcidin,ceruloplasmin, haptoglobin, orosomucoid, alpha 1-antitrypsin, alpha1-antichymotrypsin, lipopolysaccharide-binding protein (LBP), albumin,transferrin (including lactoferrin), transthyretin, retinol-bindingprotein, antithrombin, transcortin, adrenocorticotropin, Urocortin,estriol, MMP-1, MMP-2, TIMP-2, MMP-3, MMP-7, MMP-9, arachidonatelipoxygenase metabolites, prostaglandins, prostacyclins, thromboxanes,leukotrienes, Catalase, Caspase-1 (NALP3 inflammasome), leptin,adiponectin, resistin, visfatin, Retinol binding protein 4 (RBP4),endotoxin, Epidermal growth factor (EGF), insulin-like growth factorbinding protein-1 (IGFBP-1), neutrophil elastase, leukocyte elastase(ELA2, neutrophil), SLPI (secretory leukocyte protease inhibitor), S100calcium binding protein B, Heat shock protein, Endothel in-1, -2,Angiopoietin-2, Calcium-binding protein, Soluble Triggering receptorexpressed on myeloid cells 1 (sTREMi), Protein-Z (vitamin K-dependentplasma glycoprotein), and Tissue factor and Platelet activating factor(PAF).

In some embodiments, any of the antibodies, or fragments thereof, (e.g.,radiolabeled and/or PEGylated), described herein, may be used to imageimmune cells independent of an immune response. This may be done usingantibodies that detect specific immune cell markers that are notindicative of an active immune response. As one example, naïve T cellsmay be imaged using any of the radiolabelled antibodies or antibodyfragments, described herein, that bind to the naïve T cell markers CD3,CD4, CD45RA, CD45RB, CD197, or CD62L. Further information on variousimmune cell types may be found in, e.g., Zhu, J., et al.,Differentiation of effector CD4 T cell populations. Annu. Rev. Immunol.,28 (2010), pp. 445-489; S. Crotty, Follicular helper CD4 T cells (TFH),Annu. Rev. Immunol., 29 (2011), pp. 621-663. Of course it would beunderstood that certain of these markers (e.g., CD3, CD4) would also beexpressed on T cells involved in an immune response and could be used astargets for imaging an immune response.

In some embodiments, any of the antibodies, or fragments thereof, (e.g.,radiolabeled and/or PEGylated), described herein may be used tonon-invasively image tumor and/or T cell markers. In some embodiments,any of the antibodies, or fragments thereof, (e.g., radiolabeled and/orPEGylated), described herein may be used to non-invasively image a tumorcell and/or a T cell. In some embodiments, the radiolabeled antibodiesor antibody fragments detect markers including, but not limited toPD-L1, PD-1, PD-2, CTLA-4, CD3, CD4, CD8, and CD28.

In some embodiments, any of the antibodies, or fragments thereof, (e.g.,radiolabeled and/or PEGylated) described herein bind to proteinsinvolved in immune checkpoint pathways. “Immune checkpoint pathways” or“immune checkpoints” are naturally existing inhibitory pathways of theimmune system that play important roles in maintaining self-toleranceand modulating the duration and level of effector output (e.g., in thecase of T cells, the levels of cytokine production, proliferation ortarget killing potential) of physiological immune responses in order tominimize damage to the tissues of the individual mounting the immuneresponse. Such pathways may, for example, downmodulate T cell activityor enhance regulatory T cell immunosuppressive activity. Examples ofimmune checkpoint pathways include, but are not limited to the PD-1pathway and the CTLA-4 pathway and the TIM3 pathway. Tumors frequentlyco-opt certain immune-checkpoint pathways as a major mechanism of immuneresistance, e.g., against T cells that are specific for tumor antigens.Furthermore, chronic antigen exposure, such as occurs in cancer, canlead to high levels of expression of immune checkpoint proteins (e.g.,PD1, PD-L1, PD-L2) by immune cells, which can induce a state of T cellexhaustion or anergy. Certain immune checkpoint proteins such as CTLA4and PD1 are highly expressed on T regulatory (T_(Reg)) cells and mayenhance their proliferation. Many tumours are highly infiltrated withT_(Reg) cells that likely suppress effector immune responses, Thus,blockade of the PD1 pathway and/or the CTLA4 pathway may enhanceantitumour immune responses by diminishing the number and/or suppressiveactivity of intratumoral T_(Reg) cells. Certain aspects of the inventionutilize the radiolabeled proteins (e.g., radiolabeled antibodies orantibody fragments) for diagnosing or monitoring a disease or condition(e.g., cancer) or the response of a disease or condition (e.g., cancer)to therapy. For example, the radiolabeled antibodies or antibodyfragments may be used to detect whether a tumor expresses an immunecheckpoint marker (e.g., an immune checkpoint protein) and/or to detectwhether a tumor contains immune cells that express an immune checkpointmarker (e.g., an immune checkpoint protein). In other embodiments, theinventive radiolabeled proteins (e.g., radiolabeled antibodies orantibody fragments) bind to an immune checkpoint modulator. In someembodiments the immune checkpoint modulator is an immune checkpointinhibitor. “Immune checkpoint inhibitor” refers to any agent thatinhibits (suppresses, reduces activity of) an immune checkpoint pathway.In some embodiments the immune checkpoint modulator is an immunecheckpoint activator. “Immune checkpoint activator” refers to any agentthat activates (stimulates, increases activity of) an immune checkpointpathway.

Immune checkpoint inhibitors, e.g., monoclonal antibodies that bind toimmune checkpoint proteins such as CTLA4, PD1, PD-L1 have shown notableefficacy in treating a variety of different cancers, including cancersthat are advanced, have failed to respond to conventionalchemotherapeutic agents, and/or have a poor prognosis, such asmetastatic melanoma (see, e.g., Pardoll, D M, The blockade of immunecheckpoints in cancer immunotherapy, Nat Rev Cancer. 2012;12(4):252-64). However, not all subjects with tumors of a particulartype may experience benefit from treatment with a given immunecheckpoint inhibitor. One or ordinary skill would appreciate that abenefit could be, e.g., stable disease rather than progressive disease,eventual reduced number and/or volume of tumor lesions, increased meansurvival, etc. Detection of immune checkpoint markers using any of themethods, described herein, may be used to determine whether or not toadminister a therapeutic and/or to select a therapeutic (e.g., fromamong multiple different therapeutic options). For example, aradiolabeled antibody or antibody fragment that binds PD-L1 can be usedto detect whether a tumor within a patient expresses PD-L1. Patientshaving a PD-L1 positive tumor may then be administered a therapeuticthat targets the PD1 pathway, e.g., a therapeutic (such as an antibody)that targets PD1 or PD-L1. A radiolabeled protein (e.g., radiolabeledantibody or antibody fragment) that binds PD1 can be used to detectwhether a tumor within a patient is positive for PD1 (e.g., due to thepresence of immune cells that express high levels of PD1). Patientshaving a PD1 positive tumor may then be administered a therapeutic agentthat targets the PD1 pathway, In some embodiments a radiolabeled protein(e.g., radiolabeled antibody or antibody fragment) that binds TIM3 canbe used to detect whether a tumor within a patient is positive for TIM3(e.g., due to the presence of immune cells that express high levels ofTIM3). Patients having a TIM3 positive tumor may then be administered atherapeutic that targets the TIM3 pathway, e.g., a therapeutic (such asan antibody) that targets TIM3. In some embodiments a radiolabeledprotein (e.g., radiolabeled antibody or antibody fragment) that bindsCTLA4 can be used to detect whether a tumor within a patient is positivefor CTLA4 (e.g., due to the presence of immune cells that express highlevels of CTLA4). Patients having a CTLA4 positive tumor may then beadministered a therapeutic that targets the CTLA4 pathway, e.g., atherapeutic (such as an antibody) that targets CTLA4. In someembodiments a subject with a tumor may be imaged with two, three, ormore radiolabeled proteins (e.g., antibodies, antibody fragments) thatbind to different immune checkpoint proteins (e.g., proteins involved indifferent immune checkpoint pathways). One or more immune checkpointpathways that are positive in the tumor are identified. The patient isthen treated with one or more agent(s) that target those immunecheckpoint pathways for which a tumor (or one or more tumor(s)) in thesubject is positive. In some embodiments, if the tumor is negative for aparticular immune checkpoint pathway or immune checkpoint protein, analternative treatment may be administered instead of an immunecheckpoint inhibitor that would target that immune checkpoint pathway orimmune checkpoint protein. Other aspects of the invention utilize theradiolabeled antibodies or antibody fragments for monitoring theresponse to a therapeutic or monitoring expression of a protein, such asan immune checkpoint inhibitor protein. For example, a radiolabeledantibody or antibody fragment, described herein, may be used to detectwhether an immune response has been generated or enhanced or suppressedat a site of interest, such as at the site of a tumor or a site ofinfection, or whether the tumor expresses an immune checkpoint protein(e.g., PD-L1). In some embodiments a radiolabeled protein that binds toan immune cell, e.g., a T cell, may be administered to a subject before,concurrently, and/or after administration of a treatment intended toenhance or inhibit an immune response. Images may be compared frombefore and after treatment to assess the effect of the treatment on theimmune response. In some embodiments, the inventive radiolabeledproteins may be used to monitor the response to a therapeutic at leastevery 1 day, at least every 5 days, at least every 10 days, at leastevery 15 days, at least every 30 days, at least every 45 days, at leastevery 60 days, at least every 120 days, at least every 180 days, atleast every 240 days or at least every year. In some embodiments asubject may be monitored for, e.g., up to 3, 6, 9 months, up to 1, 2, 5,years, or more. In some embodiments, a therapeutic agent that targets animmune checkpoint inhibitor pathway is a monoclonal antibody. In someembodiments, the monoclonal antibody is a chimeric, humanized, or humanmonoclonal antibody. In some embodiments, the antibody is an IgGantibody, e.g., an IgG1 or IgG4 antibody. In some embodiments, atherapeutic agent that targets the CTLA4 pathway is a monoclonalantibody that binds to CTLA4, such as ipilimumab (Yervoy) ortremilimumab. In some embodiments, a therapeutic agent that targets thePD1 pathway is a monoclonal antibody that binds to PD1, such asnivolumab (a fully human IgG4 monoclonal antibody), pidilizumab (alsoknown as CT-011, a humanized IgG1 monoclonal antibody), or pembrolizumab(Keytruda, formerly lambrolizumab; also known as MK-3475), a humanizedIgG4 monoclonal antibody), or MEDI0680 (AMP-514, a humanized IgG4mAbagainst PD-1). In some embodiments, a therapeutic agent that targets thePD1 pathway is a monoclonal antibody that binds to PD-L1 such asBMS-936559 (a fully human IgG4 monoclonal antibody), MPDL3280A (humanmonoclonal, Genentech), MSB0010718C (Merck Serono), or MEDI4736. In someembodiments, a therapeutic agent that targets the PD1 pathway is amonoclonal antibody that binds to PD-L2. In some embodiments, atherapeutic agent that targets the PD1 pathway is a recombinant fusionprotein comprising extracellular domain of PD-L2 such as AMP-224. Avariety of PD1 pathway inhibitors, e.g., antibodies that bind to PD-1,PD-L1, or PD-L2 are described in U.S. Pat. Pub. No. 20040213795,20110195068, 20120039906, 20120114649, 20130095098, 20130108651,20130109843, 20130237580, and 20130291136, all of which are incorporatedby reference herein.

In some embodiments, the subject suffers from a solid tumor. In someembodiments, the subject suffers from melanoma, renal cell carcinoma,non-small-cell lung cancer, ovarian cancer, brain cancer (e.g.,glioblastoma), lymphoma (e.g., Non-Hodgkin lymphoma), hepatocellular,esophageal, breast (e.g., triple negative breast cancer), multiplemyeloma, or pancreatic cancer. In some embodiments, the subject has ametastatic cancer, stage III cancer, or stage IV cancer.

It would be understood that the immune checkpoint inhibitor could beadministered as a single agent or in combination with one or more otheranti-tumor agents.

Properties

In some embodiments, any of the antibodies, or fragments thereof, (e.g.,radiolabeled and/or PEGylated), described herein, are capable ofreaching their targets and are cleared quickly from the circulation.Whole antibodies and their fragments have different characteristics thatdetermine their targeting properties, such as how quickly they reach thetarget antigen and clear from the blood, which organ clears the antibodyfrom the blood, penetration into the tumor and amount of the injectedradiolabeled antibody or antibody fragment binding to the target. Onceantibodies target their respective antigens, they generally bind withhigh avidity, which in turn determines their tumor residence time,whereas the unbound antibody is processed by various organs in the bodyand eventually degraded and excreted. Whole IgG, which is the principalantibody form used, clears very slowly from the blood, requiring severaldays before a sufficient amount leaves the circulation to allow thespecific concentration taken into the tumor to be distinguished fromblood and adjacent tissue radioactivity. Its slow clearance is in partowing to its large size (approximately 150,000 Da) that impedes itsextravasation, resulting in a slow tumor accretion. As the molecularsize of an antibody is reduced from a divalent F(ab′)2 fragment(approximately 100,000 Da) to the monovalent binding Fab fragment(approximately 50,000 Da), there is a progressively faster clearancefrom the blood. Molecular engineering has enabled the formation of evensmaller antibody structures, such as scFv (approximately 25,000 Da),which are cleared even more rapidly from the blood. See Goldenberg D. M.et al., “Novel radiolabeled antibody conjugates.” Oncogene. 2007, 26,3734-3744; the entire contents of which are hereby incorporated byreference. Accordingly, in some embodiments any of the antibodies, orfragments thereof, (e.g., radiolabeled and/or PEGylated), describedherein, have a molecular weight of less than 60 kDa, less than 55 kDa,less than 50 kDa, less than 45 kDa, less than 40 kDa, less than 35 kDa,less than 30 kDa, less than 25 kDa less than 20 kDa, less than 15 kDa,less than 10 kDa, or less than 5 kDa. In other embodiments theradiolabelled proteins (e.g., antibodies or antibody fragments),described herein, have a molecular weight ranging from 5 kDa-15 kDa,from 5 kDa-20 kDa, from 5 kDa-25 kDa, from 5 kDa-30 kDa, from 5 kDa-35kDa, from 5 kDa-40 kDa, from 5 kDa-45 kDa, from 5 kDa-55 kDa, from 5kDa-60 kDa, from 15 kDa-20 kDa, from 15 kDa-25 kDa, from 15 kDa-30 kDa,from 15 kDa-35 kDa, from 15 kDa-40 kDa, from 15 kDa-45 kDa, from 15kDa-50 kDa, from 15 kDa-55 kDa, from 15 kDa-60 kDa, from 25 kDa-35 kDa,from 25 kDa-45 kDa, from 25 kDa-55 kDa, from 25 kDa-60 kDa, from 35kDa-45 kDa, from 35 kDa-55 kDa, from 35 kDa-60 kDa, from 45 kDa-55 kDa,from 45 kDa-60 kDa, or from 50 kDa-60 kDa. In yet other embodiments, anyof the antibodies, or fragments thereof, (e.g., radiolabeled and/orPEGylated), described herein, are expediently cleared from thecirculation following injection into a patient. In some embodiments, atleast 95% of the radiolabelled proteins (e.g., antibodies or antibodyfragments) are cleared from the blood within 20 minutes, within 30minutes, within 40 minutes, within 60 minutes, within 80 minutes, within30 minutes, within 40 minutes, within 60 minutes, within 90 minutes,within 2 hours, within 3 hours, within 4 hours, within 6 hours, within 8hours, within 10 hours or within 12 hours. In other embodiments, atleast 95% of the radiolabelled proteins (e.g., antibodies or antibodyfragments) are cleared from the body within 20 minutes, within 30minutes, within 40 minutes, within 60 minutes, within 80 minutes, within30 minutes, within 40 minutes, within 60 minutes, within 90 minutes,within 2 hours, within 3 hours, within 4 hours, within 6 hours, within 8hours, within 10 hours or within 12 hours.

Hydrophilic Polymers

Some aspects of the disclosure are related to the use hydrophilicpolymers and methods of conjugating hydrophilic polymers to an antibody(e.g., a single domain antibody). As used herein, a “hydrophilicpolymer” refers to a molecule (e.g., a macromolecule) comprised of threeor more repeating units that contain polar or charged functional groups,rendering them soluble in water. In some embodiments, the hydrophilicpolymer is soluble to at least 50 mg/mL in water, to at least 100 mg/mLin water, to at least 150 mg/mL in water, to at least 200 mg/mL inwater, to at least 250 mg/mL in water, to at least 300 mg/mL in water,to at least 400 mg/mL in water, to at least 500 mg/mL in water, to atleast 600 mg/mL in water, to at least 700 mg/mL in water, to at least800 mg/mL in water, to at least 900 mg/mL in water, or at least 1000mg/mL in water at 20° C. In some embodiments, the hydrophilic polymer isa linear polymer. In some embodiments, the hydrophilic polymer is abranched polymer, which may be configured in any number of ways. In someembodiments, the hydrophilic polymer is a suitable size and/or has asuitable linear or branched structure for conferring improved propertiesto any of the antibodies provided herein. In some embodiments, thehydrophilic polymer is from 5 kDa to 1000 kDa in size. In someembodiments, the polymer is at least 5 kDa, at least 10 kDa, at least 15kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 40 kDa,at least 50 kDa, at least 70 kDa, at least 100 kDa, at least 150 kDa, atleast 200 kDa, at least 300 kDa, at least 400 kDa, at least 500 kDa, atleast 600 kDa, at least 700 kDa, at least 800 kDa, at least 900 kDa, orat least 1000 kDa in size.

In some embodiments, the hydrophilic polymer useful in the presentinvention is synthetic polymer. In certain embodiments, the hydrophilicpolymer is not a polypeptide, polynucleotide, or polysaccharide.Examples of hydrophilic synthetic polymers include, without limitationpolyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), polyvinylalcohol (PVA), polyacrylic acid (PAA), polyacrylamide,N-(2-hydroxypropyl) methacrylamide (HPMA), divinyl ether-maleicanhydride (DIVEMA), polyoxazoline, polyphosphoester (PPE),polyethyleneimine (PEI), and polyphosphazene. However, it should beappreciated that other synthetic polymers may be used and are consideredto be within the scope of this disclosure.

In some embodiments, any of the hydrophilic polymers provided herein arenatural polymers. Examples of natural polymers include, withoutlimitation, polysaccharides, xanthan gum, pectins, chitosan derivatives,dextran, carrageenan, guar gum, cellulose ethers (e.g., Sodium CMC, HPC,and HPMC), hyaluronic acid (HA), albumin, and starch or starch basedderivatives. However, it should be appreciated that other syntheticpolymers may be used and are within the scope of this disclosure.Additional hydrophilic polymers would be apparent to the skilled artisanand are within the scope of this disclosure. For example, exemplaryhydrophilic polymers have been described previously in Veeran, G. K., etal., “Water Soluble Polymers for Pharmaceutical Applications” Polymers2011, 3, 1972-2009; the entire contents of which are incorporated byreference herein. It should be appreciated that, in some embodiments,PEGylation also refers to the addition of any hydrophilic polymer, forexample any of the hydrophilic polymers provided herein.

Chelating Moieties

Some aspects of the disclosure provide chelating moieties and methodsfor conjugating chelating moieties to antibodies (e.g., single domainantibodies). As used here a “chelating moiety” is an agent whosemolecules can form several bonds to a single metal ion. In someembodiments, the chelating moiety is an agent that binds to aradionuclide. In some embodiments, the chelating moiety is an agent thatbinds to rubidium-82, copper-61, copper-62, copper-64, yttrium-86,gallium-68, zirconium-89. However, it should be appreciated that thechelating moiety may be an agent that binds to additional radioactivemetals. Exemplary chelating moieties include, without limitation,1,4,7-triazacyclononane-triacetic acid (NOTA),1,4,7,10-tetraazacyclododecane-tetraacetic acid (DOTA),triazacyclononane-phosphinate (TRAP), or desferrioxamine (DFO). However,it should be appreciated that additional chelating moieties may be usedand are within the scope of this invention. Exemplary metallicradionuclides and chelators have been described in the art and arewithin the scope of this disclosure. For example, exemplary metallicradionuclides and chelators have been described in Liu X., et al., “ABrief Review of Chelators for Radiolabeling Oligomers” Materials, 2010,3, 3204-3217, the entire contents of which are incorporated by referenceherein.

Chemistry Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various stereoisomeric forms, e.g., enantiomersand/or diastereomers. For example, the compounds described herein can bein the form of an individual enantiomer, diastereomer or geometricisomer, or can be in the form of a mixture of stereoisomers, includingracemic mixtures and mixtures enriched in one or more stereoisomer.Isomers can be isolated from mixtures by methods known to those skilledin the art, including chiral high pressure liquid chromatography (HPLC)and the formation and crystallization of chiral salts; or preferredisomers can be prepared by asymmetric syntheses. See, for example,Jacques et al., Enantiomers, Racemates and Resolutions (WileyInterscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977);Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, N Y,1962); and Wilen, S. H. Tables of Resolving Agents and OpticalResolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, NotreDame, Ind. 1972). The invention additionally encompasses compounds asindividual isomers substantially free of other isomers, andalternatively, as mixtures of various isomers.

In a formula, --- is absent, a coordination bond between a ligand and ametal, or a single bond.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

The term “aliphatic,” as used herein, includes both saturated andunsaturated, nonaromatic, straight chain (i.e., unbranched), branched,acyclic, and cyclic (i.e., carbocyclic) hydrocarbons, which areoptionally substituted with one or more functional groups. As will beappreciated by one of ordinary skill in the art, “aliphatic” is intendedherein to include, but is not limited to, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as usedherein, the term “alkyl” includes straight, branched and cyclic alkylgroups. An analogous convention applies to other generic terms such as“alkenyl,” “alkynyl,” and the like. Furthermore, as used herein, theterms “alkyl,” “alkenyl,” “alkynyl,” and the like encompass bothsubstituted and unsubstituted groups. In certain embodiments, as usedherein, “aliphatic” is used to indicate those aliphatic groups (cyclic,acyclic, substituted, unsubstituted, branched or unbranched) having 1-20carbon atoms (C₁₋₂₀ aliphatic). In certain embodiments, the aliphaticgroup has 1-10 carbon atoms (C₁₋₁₀ aliphatic). In certain embodiments,the aliphatic group has 1-6 carbon atoms (C₁₋₆ aliphatic). In certainembodiments, the aliphatic group has 1-5 carbon atoms (C₁₋₅ aliphatic).In certain embodiments, the aliphatic group has 1-4 carbon atoms (C₁₋₄aliphatic). In certain embodiments, the aliphatic group has 1-3 carbonatoms (C₁₋₃ aliphatic). In certain embodiments, the aliphatic group has1-2 carbon atoms (C₁₋₂ aliphatic). Aliphatic group substituents include,but are not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety.

The term “heteroaliphatic”, as used herein, refers to aliphatic moietiesthat contain one or more oxygen, sulfur, nitrogen, phosphorus, orsilicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moietiesmay be branched, unbranched, cyclic or acyclic and include saturated andunsaturated heterocycles such as morpholino, pyrrolidinyl, etc. Incertain embodiments, heteroaliphatic moieties are substituted byindependent replacement of one or more of the hydrogen atoms thereonwith one or more moieties including, but not limited to aliphatic;heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I; —OH; —NO₂; —CN; —CF₃;—CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x);—CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x); —OCON(R_(x))₂;—N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x), wherein each occurrence ofR_(x) independently includes, but is not limited to, aliphatic,heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,wherein any of the aliphatic, heteroaliphatic, arylalkyl, orheteroarylalkyl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, cyclic or acyclic,and wherein any of the aryl or heteroaryl substituents described aboveand herein may be substituted or unsubstituted. Additional examples ofgenerally applicable substitutents are illustrated by the specificembodiments shown in the Examples that are described herein.

The term “alkyl,” as used herein, refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom. In some embodiments, the alkyl group employed in theinvention contains 1-20 carbon atoms (C₁₋₂₀alkyl). In anotherembodiment, the alkyl group employed contains 1-15 carbon atoms(C₁₋₁₅alkyl). In another embodiment, the alkyl group employed contains1-10 carbon atoms (C₁₋₁₀alkyl). In another embodiment, the alkyl groupemployed contains 1-8 carbon atoms (C₁₋₈alkyl). In another embodiment,the alkyl group employed contains 1-6 carbon atoms (C₁₋₆alkyl). Inanother embodiment, the alkyl group employed contains 1-5 carbon atoms(C₁₋₅alkyl). In another embodiment, the alkyl group employed contains1-4 carbon atoms (C₁₋₄alkyl). In another embodiment, the alkyl groupemployed contains 1-3 carbon atoms (C₁₋₃alkyl). In another embodiment,the alkyl group employed contains 1-2 carbon atoms (C₁₋₂alkyl). Examplesof alkyl radicals include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl,iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl,n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like, which maybear one or more substituents. Alkyl group substituents include, but arenot limited to, any of the substituents described herein, that result inthe formation of a stable moiety. The term “alkylene,” as used herein,refers to a biradical derived from an alkyl group, as defined herein, byremoval of two hydrogen atoms. Alkylene groups may be cyclic or acyclic,branched or unbranched, substituted or unsubstituted. Alkylene groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety.

The term “alkenyl,” as used herein, denotes a monovalent group derivedfrom a straight- or branched-chain hydrocarbon moiety having at leastone carbon-carbon double bond by the removal of a single hydrogen atom.In certain embodiments, the alkenyl group employed in the inventioncontains 2-20 carbon atoms (C₂₋₂₀alkenyl). In some embodiments, thealkenyl group employed in the invention contains 2-15 carbon atoms(C₂₋₁₅alkenyl). In another embodiment, the alkenyl group employedcontains 2-10 carbon atoms (C₂₋₁₀alkenyl). In still other embodiments,the alkenyl group contains 2-8 carbon atoms (C₂₋₈alkenyl). In yet otherembodiments, the alkenyl group contains 2-6 carbons (C₂₋₆alkenyl). Inyet other embodiments, the alkenyl group contains 2-5 carbons(C₂₋₅alkenyl). In yet other embodiments, the alkenyl group contains 2-4carbons (C₂₋₄alkenyl). In yet other embodiments, the alkenyl groupcontains 2-3 carbons (C₂₋₃alkenyl). In yet other embodiments, thealkenyl group contains 2 carbons (C₂alkenyl). Alkenyl groups include,for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and thelike, which may bear one or more substituents. Alkenyl groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety. Theterm “alkenylene,” as used herein, refers to a biradical derived from analkenyl group, as defined herein, by removal of two hydrogen atoms.Alkenylene groups may be cyclic or acyclic, branched or unbranched,substituted or unsubstituted. Alkenylene group substituents include, butare not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety.

The term “alkynyl,” as used herein, refers to a monovalent group derivedfrom a straight- or branched-chain hydrocarbon having at least onecarbon-carbon triple bond by the removal of a single hydrogen atom. Incertain embodiments, the alkynyl group employed in the inventioncontains 2-20 carbon atoms (C₂₋₂₀alkynyl). In some embodiments, thealkynyl group employed in the invention contains 2-15 carbon atoms(C₂₋₁₅alkynyl). In another embodiment, the alkynyl group employedcontains 2-10 carbon atoms (C₂₋₁₀alkynyl). In still other embodiments,the alkynyl group contains 2-8 carbon atoms (C₂₋₈alkynyl). In stillother embodiments, the alkynyl group contains 2-6 carbon atoms(C₂₋₆alkynyl). In still other embodiments, the alkynyl group contains2-5 carbon atoms (C₂₋₅alkynyl). In still other embodiments, the alkynylgroup contains 2-4 carbon atoms (C₂₋₄alkynyl). In still otherembodiments, the alkynyl group contains 2-3 carbon atoms (C₂₋₃alkynyl).In still other embodiments, the alkynyl group contains 2 carbon atoms(C₂alkynyl). Representative alkynyl groups include, but are not limitedto, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like, which maybear one or more substituents. Alkynyl group substituents include, butare not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety. The term “alkynylene,” asused herein, refers to a biradical derived from an alkynylene group, asdefined herein, by removal of two hydrogen atoms. Alkynylene groups maybe cyclic or acyclic, branched or unbranched, substituted orunsubstituted. Alkynylene group substituents include, but are notlimited to, any of the substituents described herein, that result in theformation of a stable moiety.

The term “heteroalkyl” refers to an alkyl group, which further includesat least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected fromoxygen, nitrogen, or sulfur within (i.e., inserted between adjacentcarbon atoms of) and/or placed at one or more terminal position(s) ofthe parent chain. In certain embodiments, a heteroalkyl group refers toa saturated group having from 1 to 10 carbon atoms and 1 or moreheteroatoms within the parent chain (“heteroC₁₋₁₀ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 9carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₉ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 8 carbon atoms and 1 or more heteroatomswithin the parent chain (“heteroC₁₋₈ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1or more heteroatoms within the parent chain (“heteroC₁₋₇ alkyl”). Insome embodiments, a heteroalkyl group is a saturated group having 1 to 6carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₆ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms withinthe parent chain (“heteroC₁₋₅ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1or 2 heteroatoms within the parent chain (“heteroC₁₋₄ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 3carbon atoms and 1 heteroatom within the parent chain (“heteroC₁₋₃alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 1 to 2 carbon atoms and 1 heteroatom within the parent chain(“heteroC₁₋₂ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 carbon atom and 1 heteroatom (“heteroC₁alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parentchain (“heteroC₂₋₆ alkyl”). Unless otherwise specified, each instance ofa heteroalkyl group is independently unsubstituted (an “unsubstitutedheteroalkyl”) or substituted (a “substituted heteroalkyl”) with one ormore substituents. In certain embodiments, the heteroalkyl group is anunsubstituted heteroC₁₋₁₀ alkyl. In certain embodiments, the heteroalkylgroup is a substituted heteroC₁₋₁₀ alkyl.

The term “heteroalkenyl” refers to an alkenyl group, which furtherincludes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms)selected from oxygen, nitrogen, or sulfur within (i.e., inserted betweenadjacent carbon atoms of) and/or placed at one or more terminalposition(s) of the parent chain. In certain embodiments, a heteroalkenylgroup refers to a group having from 2 to 10 carbon atoms, at least onedouble bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₁₀ alkenyl”). In some embodiments, a heteroalkenyl group has2 to 9 carbon atoms at least one double bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₉ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 8 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 7 carbonatoms, at least one double bond, and 1 or more heteroatoms within theparent chain (“heteroC₂₋₇ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 6 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbonatoms, at least one double bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 4 carbon atoms, at least one double bond,and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkenyl”).In some embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, atleast one double bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkenyl”). In some embodiments, a heteroalkenyl group has 2to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkenyl”). Unless otherwisespecified, each instance of a heteroalkenyl group is independentlyunsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a“substituted heteroalkenyl”) with one or more substituents. In certainembodiments, the heteroalkenyl group is an unsubstituted heteroC₂₋₁₀alkenyl. In certain embodiments, the heteroalkenyl group is asubstituted heteroC₂₋₁₀ alkenyl.

The term “heteroalkynyl” refers to an alkynyl group, which furtherincludes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms)selected from oxygen, nitrogen, or sulfur within (i.e., inserted betweenadjacent carbon atoms of) and/or placed at one or more terminalposition(s) of the parent chain. In certain embodiments, a heteroalkynylgroup refers to a group having from 2 to 10 carbon atoms, at least onetriple bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₁₀ alkynyl”). In some embodiments, a heteroalkynyl group has2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₉ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 7 carbonatoms, at least one triple bond, and 1 or more heteroatoms within theparent chain (“heteroC₂₋₇ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbonatoms, at least one triple bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond,and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkynyl”).In some embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, atleast one triple bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkynyl”). In some embodiments, a heteroalkynyl group has 2to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkynyl”). Unless otherwisespecified, each instance of a heteroalkynyl group is independentlyunsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a“substituted heteroalkynyl”) with one or more substituents. In certainembodiments, the heteroalkynyl group is an unsubstituted heteroC₂₋₁₀alkynyl. In certain embodiments, the heteroalkynyl group is asubstituted heteroC₂₋₁₀ alkynyl.

The term “carbocyclyl” or “carbocyclic” refers to a radical of anon-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbonatoms (“C₃₋₁₄ carbocyclyl”) and zero heteroatoms in the non-aromaticring system. In some embodiments, a carbocyclyl group has 3 to 10 ringcarbon atoms (“C₃₋₁₀ carbocyclyl”). In some embodiments, a carbocyclylgroup has 3 to 8 ring carbon atoms (“C₃₋₈ carbocyclyl”). In someembodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C₃₋₇carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ringcarbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, a carbocyclylgroup has 4 to 6 ring carbon atoms (“C₄₋₆ carbocyclyl”). In someembodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C₅₋₆carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groupsinclude, without limitation, cyclopropyl (C₃), cyclopropenyl (C₃),cyclobutyl (C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl(C₅), cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and thelike. Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing afused, bridged or spiro ring system such as a bicyclic system (“bicycliccarbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can besaturated or can contain one or more carbon-carbon double or triplebonds. “Carbocyclyl” also includes ring systems wherein the carbocyclylring, as defined above, is fused with one or more aryl or heteroarylgroups wherein the point of attachment is on the carbocyclyl ring, andin such instances, the number of carbons continue to designate thenumber of carbons in the carbocyclic ring system. Unless otherwisespecified, each instance of a carbocyclyl group is independentlyunsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is an unsubstituted C₃₋₁₄carbocyclyl. In certain embodiments, the carbocyclyl group is asubstituted C₃₋₁₄ carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 14 ring carbon atoms (“C₃₋₁₄cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 10 ringcarbon atoms (“C₃₋₁₀ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 8 ring carbon atoms (“C₃₋₈ cycloalkyl”). In someembodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C₃₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ringcarbon atoms (“C₄₋₆ cycloalkyl”). In some embodiments, a cycloalkylgroup has 5 to 6 ring carbon atoms (“C₅₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C₅₋₁₀cycloalkyl”). Examples of C₅₋₆ cycloalkyl groups include cyclopentyl(C₅) and cyclohexyl (C₅). Examples of C₃₋₆ cycloalkyl groups include theaforementioned C₅₋₆ cycloalkyl groups as well as cyclopropyl (C₃) andcyclobutyl (C₄). Examples of C₃₋₈ cycloalkyl groups include theaforementioned C₃₋₆ cycloalkyl groups as well as cycloheptyl (C₇) andcyclooctyl (C₈). Unless otherwise specified, each instance of acycloalkyl group is independently unsubstituted (an “unsubstitutedcycloalkyl”) or substituted (a “substituted cycloalkyl”) with one ormore substituents. In certain embodiments, the cycloalkyl group is anunsubstituted C₃₋₁₄ cycloalkyl. In certain embodiments, the cycloalkylgroup is a substituted C₃₋₁₄ cycloalkyl.

The term “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to14-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or polycyclic (e.g., a fused, bridged or spiro ring system such as abicyclic system (“bicyclic heterocyclyl”) or tricyclic system(“tricyclic heterocyclyl”)), and can be saturated or can contain one ormore carbon-carbon double or triple bonds. Heterocyclyl polycyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.In certain embodiments, the heterocyclyl group is a substituted 3-14membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-8 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl groupis a 5-6 membered non-aromatic ring system having ring carbon atoms and1-4 ring heteroatoms, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azirdinyl, oxiranyl, and thiiranyl.Exemplary 4-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azetidinyl, oxetanyl, and thietanyl.Exemplary 5-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl,and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining 2 heteroatoms include, without limitation, dioxolanyl,oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groupscontaining 3 heteroatoms include, without limitation, triazolinyl,oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclylgroups containing 1 heteroatom include, without limitation, piperidinyl,tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-memberedheterocyclyl groups containing 2 heteroatoms include, withoutlimitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary6-membered heterocyclyl groups containing 2 heteroatoms include, withoutlimitation, triazinanyl. Exemplary 7-membered heterocyclyl groupscontaining 1 heteroatom include, without limitation, azepanyl, oxepanyland thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1heteroatom include, without limitation, azocanyl, oxecanyl andthiocanyl. Exemplary bicyclic heterocyclyl groups include, withoutlimitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl,tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl,octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g.,bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or14π electrons shared in a cyclic array) having 6-14 ring carbon atomsand zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C₁₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systems whereinthe aryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the radical or point of attachment is onthe aryl ring, and in such instances, the number of carbon atomscontinue to designate the number of carbon atoms in the aryl ringsystem. Unless otherwise specified, each instance of an aryl group isindependently unsubstituted (an “unsubstituted aryl”) or substituted (a“substituted aryl”) with one or more substituents. In certainembodiments, the aryl group is an unsubstituted C₆₋₁₄ aryl. In certainembodiments, the aryl group is a substituted C₆₋₁₄ aryl.

The term “heteroaryl” refers to a radical of a 5-14 membered monocyclicor polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system(e.g., having 6, 10, or 14π electrons shared in a cyclic array) havingring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ringsystem, wherein each heteroatom is independently selected from nitrogen,oxygen, and sulfur (“5-14 membered heteroaryl”). In heteroaryl groupsthat contain one or more nitrogen atoms, the point of attachment can bea carbon or nitrogen atom, as valency permits. Heteroaryl polycyclicring systems can include one or more heteroatoms in one or both rings.“Heteroaryl” includes ring systems wherein the heteroaryl ring, asdefined above, is fused with one or more carbocyclyl or heterocyclylgroups wherein the point of attachment is on the heteroaryl ring, and insuch instances, the number of ring members continue to designate thenumber of ring members in the heteroaryl ring system. “Heteroaryl” alsoincludes ring systems wherein the heteroaryl ring, as defined above, isfused with one or more aryl groups wherein the point of attachment iseither on the aryl or heteroaryl ring, and in such instances, the numberof ring members designates the number of ring members in the fusedpolycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groupswherein one ring does not contain a heteroatom (e.g., indolyl,quinolinyl, carbazolyl, and the like) the point of attachment can be oneither ring, i.e., either the ring bearing a heteroatom (e.g.,2-indolyl) or the ring that does not contain a heteroatom (e.g.,5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently unsubstituted (an “unsubstituted heteroaryl”) orsubstituted (a “substituted heteroaryl”) with one or more substituents.In certain embodiments, the heteroaryl group is an unsubstituted 5-14membered heteroaryl. In certain embodiments, the heteroaryl group is asubstituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include,without limitation, pyrrolyl, furanyl, and thiophenyl. Exemplary5-membered heteroaryl groups containing 2 heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing 3heteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4heteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing 1 heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, andpyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4heteroatoms include, without limitation, triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing 1heteroatom include, without limitation, azepinyl, oxepinyl, andthiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, withoutlimitation, indolyl, isoindolyl, indazolyl, benzotriazolyl,benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl,benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl,benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, andpurinyl. Exemplary 6,6-bicyclic heteroaryl groups include, withoutlimitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplarytricyclic heteroaryl groups include, without limitation,phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl,phenoxazinyl and phenazinyl.

The term “unsaturated” or “partially unsaturated” refers to a moietythat includes at least one double or triple bond.

Affixing the suffix “-ene” to a group indicates the group is a divalentmoiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene isthe divalent moiety of alkenyl, alkynylene is the divalent moiety ofalkynyl, heteroalkylene is the divalent moiety of heteroalkyl,heteroalkenylene is the divalent moiety of heteroalkenyl,heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclyleneis the divalent moiety of carbocyclyl, heterocyclylene is the divalentmoiety of heterocyclyl, arylene is the divalent moiety of aryl, andheteroarylene is the divalent moiety of heteroaryl.

A group is optionally substituted unless expressly provided otherwise.In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl groups are optionally substituted. “Optionally substituted”refers to a group which may be substituted or unsubstituted (e.g.,“substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted”alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” heteroalkyl, “substituted” or “unsubstituted”heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl,“substituted” or “unsubstituted” carbocyclyl, “substituted” or“unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or“substituted” or “unsubstituted” heteroaryl group). In general, the term“substituted” means that at least one hydrogen present on a group isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound, e.g., a compound which doesnot spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction. Unless otherwise indicated,a “substituted” group has a substituent at one or more substitutablepositions of the group, and when more than one position in any givenstructure is substituted, the substituent is either the same ordifferent at each position. The term “substituted” is contemplated toinclude substitution with all permissible substituents of organiccompounds, and includes any of the substituents described herein thatresults in the formation of a stable compound. The present inventioncontemplates any and all such combinations in order to arrive at astable compound. For purposes of this invention, heteroatoms such asnitrogen may have hydrogen substituents and/or any suitable substituentas described herein which satisfy the valencies of the heteroatoms andresults in the formation of a stable moiety. The invention is notintended to be limited in any manner by the exemplary substituentsdescribed herein.

The term “halo” or “halogen” refers to fluorine (fluoro, —F), chlorine(chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

The term “amino” refers to the group —NH₂. The term “substituted amino,”by extension, refers to a monosubstituted amino, a disubstituted amino,or a trisubstituted amino. In certain embodiments, the “substitutedamino” is a monosubstituted amino or a disubstituted amino group.

The term “monosubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith one hydrogen and one group other than hydrogen, and includes groupsselected from —NH(R^(bb)), —NHC(═O)R^(aa), —NHCO₂R^(aa),—NHC(═O)N(R^(bb))₂, —NHC(═NR^(bb))N(R^(bb))₂, —NHSO₂R^(aa),—NHP(═O)(OR^(cc))₂, and —NHP(═O)(NR^(bb))₂, wherein R^(aa), R^(bb) andR^(cc) are as defined herein, and wherein R^(bb) of the group—NH(R^(bb)) is not hydrogen.

The term “disubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith two groups other than hydrogen, and includes groups selected from—N(R^(bb))₂, —NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa),—NR^(bb)C(═O)N(R^(bb))₂, —NR^(bb)C(═NR^(bb))N(R^(bb))₂,—NR^(bb)SO₂R^(aa), —NR^(bb)P(═O)(OR^(cc))₂, and —NR^(bb)P(═O)(NR^(bb))₂,wherein R^(aa), R^(bb), and R^(cc) are as defined herein, with theproviso that the nitrogen atom directly attached to the parent moleculeis not substituted with hydrogen.

The term “trisubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith three groups, and includes groups selected from —N(R^(bb))₃ and—N(R^(bb))₃ ⁺X⁻, wherein R^(bb) and X⁻ are as defined herein.

In certain embodiments, the substituent present on the nitrogen atom isan nitrogen protecting group (also referred to herein as an “aminoprotecting group”). Nitrogen protecting groups include, but are notlimited to, —OH, —OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂,—CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and R^(dd) are asdefined herein. Nitrogen protecting groups are well known in the art andinclude those described in detail in Protecting Groups in OrganicSynthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley &Sons, 1999, incorporated herein by reference.

For example, nitrogen protecting groups such as amide groups (e.g.,—C(═O)R^(aa)) include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g.,—C(═O)OR^(aa)) include, but are not limited to, methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc),vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallylcarbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate(Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g.,—S(═O)₂R^(aa)) include, but are not limited to, p-toluenesulfonamide(Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide(Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to herein as an “hydroxylprotecting group”). Oxygen protecting groups include, but are notlimited to, —R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa),—CO₂R^(aa), —C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethylcarbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate(Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc),isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate(BOC or Boc), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzylcarbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate,p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-naphthylcarbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate,4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, o-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

In certain embodiments, the substituent present on an sulfur atom is asulfur protecting group (also referred to as a “thiol protectinggroup”). Sulfur protecting groups include, but are not limited to,—R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Sulfur protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

The terms “aminooxy,” or “aminooxy group,” are used interchangeablyherein and refer to functional groups having the general formula:

wherein R³¹ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, optionally substitutedheteroaryl, or optionally substituted heterocyclyl. In some embodiments,R³¹ is an amino acid, wherein the point of attachment for the oxygen ison the side chain of the amino acid. In certain embodiments, the aminoacid is within a polypeptide.

The term “tautomers” or “tautomeric” refers to two or moreinterconvertable compounds resulting from at least one formal migrationof a hydrogen atom and at least one change in valency (e.g., a singlebond to a double bond, a triple bond to a single bond, or vice versa).The exact ratio of the tautomers depends on several factors, includingtemperature, solvent, and pH. Tautomerizations (i.e., the reactionproviding a tautomeric pair) may catalyzed by acid or base. Exemplarytautomerizations include keto-to-enol, amide-to-imide, lactam-to-lactim,enamine-to-imine, and enamine-to-(a different enamine) tautomerizations.

It is also to be understood that compounds that have the same molecularformula but differ in the nature or sequence of bonding of their atomsor the arrangement of their atoms in space are termed “isomers”. Isomersthat differ in the arrangement of their atoms in space are termed“stereoisomers”.

Stereoisomers that are not mirror images of one another are termed“diastereomers” and those that are non-superimposable mirror images ofeach other are termed “enantiomers”. When a compound has an asymmetriccenter, for example, it is bonded to four different groups, a pair ofenantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is described by theR- and S-sequencing rules of Cahn and Prelog, or by the manner in whichthe molecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e., as (+) or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a “racemic mixture”.

The terms “carbonyl,” or “carbonyl group,” are used interchangeablyherein and refer to functional groups composed of a carbon atomdouble-bonded to any oxygen atom. Carbonyls have the general formula:

wherein each of R³² and R³³ independently represents hydroxyl,optionally substituted amino, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl,optionally substituted heteroaryl, or optionally substitutedheterocyclyl. Examples of carbonyls include, but are not limited to,aldehydes, ketones, carboxylic acids, esters, amides, enones, acylhalides, acid anhydrides, and imides. In some embodiments, acarbonyl-containing compound refers to a compound having an aldehydegroup, or a compound capable of forming an aldehyde group throughisomerization. For example, in some embodiments, certain sugars (e.g.,reducing sugars) such as glucose, form aldehydes through isomerization.A sugar is classified as a reducing sugar if it has an open-chain formwith an aldehyde group or a free hemiacetal group. Monosaccharides whichcontain an aldehyde group are known as aldoses, and those with a ketonegroup are known as ketoses. The aldehyde can be oxidized via a redoxreaction in which another compound is reduced. Thus, a reducing sugar isone that is capable of reducing certain chemicals. Sugars with ketonegroups in their open chain form are capable of isomerizing via a seriesof tautomeric shifts to produce an aldehyde group in solution.Therefore, ketone-bearing sugars like fructose are considered reducingsugars but it is the isomer containing an aldehyde group which isreducing since ketones cannot be oxidized without decomposition of thesugar. This type of isomerization is catalyzed by the base present insolutions which test for the presence of aldehydes.

The term “hydrazide,” as used herein, refers to functional groups havingthe general formula:

wherein R³⁴ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, optionally substitutedheteroaryl, or optionally substituted heterocyclyl. In some embodiments,R³⁴ is an amino acid, wherein the point of attachment for the oxygen ison the side chain of the amino acid. In certain embodiments, the aminoacid is within a polypeptide.

The term “hydrazone,” as used herein, refers to compound having thegeneral formula:

wherein each of R³⁵, R³⁶, and R³⁷ is independently optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, optionally substituted heteroaryl, oroptionally substituted heterocyclyl. The term “hydrazone linkage,” asused herein, refers to the formula:

Hydrzones can be prepared from, for example, joining of a compoundcomprising a hydrazide group and a compound comprising a carbonyl.

The term “acyl,” as used herein, is a subset of a substituted alkylgroup, and refers to a group having the general formula —C(═O)R^(A),—C(═O)OR^(A), —C(═O)—O—C(═O)R^(A), —C(═O)SR^(A), or —C(═O)N(R^(A))₂,wherein each instance of R^(A) is independently hydrogen, optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, optionally substituted heteroaryl, oroptionally substituted heterocyclyl. Exemplary acyl groups includealdehydes (—CHO), carboxylic acids (—CO₂H), ketones, acyl halides,esters, amides, imines, carbonates, carbamates, and ureas. Acylsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety.

The term “azide” or “azido,” as used herein, refers to a group of theformula (—N₃).

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various stereoisomeric forms, e.g., enantiomersand/or diastereomers. For example, the compounds described herein can bein the form of an individual enantiomer, diastereomer or geometricisomer, or can be in the form of a mixture of stereoisomers, includingracemic mixtures and mixtures enriched in one or more stereoisomer.Isomers can be isolated from mixtures by methods known to those skilledin the art, including chiral high pressure liquid chromatography (HPLC)and the formation and crystallization of chiral salts; or preferredisomers can be prepared by asymmetric syntheses. See, for example,Jacques et al., Enantiomers, Racemates and Resolutions (WileyInterscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977);Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, N Y,1962); and Wilen, S. H. Tables of Resolving Agents and OpticalResolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, NotreDame, Ind. 1972). The invention additionally encompasses compounds asindividual isomers substantially free of other isomers, andalternatively, as mixtures of various isomers.

In a formula, --- is absent, a coordination bond between a ligand and ametal, or a single bond.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

The term “aliphatic,” as used herein, includes both saturated andunsaturated, nonaromatic, straight chain (i.e., unbranched), branched,acyclic, and cyclic (i.e., carbocyclic) hydrocarbons, which areoptionally substituted with one or more functional groups. As will beappreciated by one of ordinary skill in the art, “aliphatic” is intendedherein to include, but is not limited to, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as usedherein, the term “alkyl” includes straight, branched and cyclic alkylgroups. An analogous convention applies to other generic terms such as“alkenyl,” “alkynyl,” and the like. Furthermore, as used herein, theterms “alkyl,” “alkenyl,” “alkynyl,” and the like encompass bothsubstituted and unsubstituted groups. In certain embodiments, as usedherein, “aliphatic” is used to indicate those aliphatic groups (cyclic,acyclic, substituted, unsubstituted, branched or unbranched) having 1-20carbon atoms (C₁₋₂₀ aliphatic). In certain embodiments, the aliphaticgroup has 1-10 carbon atoms (C₁₋₁₀ aliphatic). In certain embodiments,the aliphatic group has 1-6 carbon atoms (C₁₋₆ aliphatic). In certainembodiments, the aliphatic group has 1-5 carbon atoms (C₁₋₅ aliphatic).In certain embodiments, the aliphatic group has 1-4 carbon atoms (C₁₋₄aliphatic). In certain embodiments, the aliphatic group has 1-3 carbonatoms (C₁₋₃ aliphatic). In certain embodiments, the aliphatic group has1-2 carbon atoms (C₁₋₂ aliphatic). Aliphatic group substituents include,but are not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety.

The term “heteroaliphatic”, as used herein, refers to aliphatic moietiesthat contain one or more oxygen, sulfur, nitrogen, phosphorus, orsilicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moietiesmay be branched, unbranched, cyclic or acyclic and include saturated andunsaturated heterocycles such as morpholino, pyrrolidinyl, etc. Incertain embodiments, heteroaliphatic moieties are substituted byindependent replacement of one or more of the hydrogen atoms thereonwith one or more moieties including, but not limited to aliphatic;heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I; —OH; —NO₂; —CN; —CF₃;—CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x);—CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x); —OCON(R_(x))₂;—N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x), wherein each occurrence ofR_(x) independently includes, but is not limited to, aliphatic,heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,wherein any of the aliphatic, heteroaliphatic, arylalkyl, orheteroarylalkyl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, cyclic or acyclic,and wherein any of the aryl or heteroaryl substituents described aboveand herein may be substituted or unsubstituted. Additional examples ofgenerally applicable substitutents are illustrated by the specificembodiments shown in the Examples that are described herein.

The term “alkyl,” as used herein, refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom. In some embodiments, the alkyl group employed in theinvention contains 1-20 carbon atoms (C₁₋₂₀alkyl). In anotherembodiment, the alkyl group employed contains 1-15 carbon atoms(C₁₋₁₅alkyl). In another embodiment, the alkyl group employed contains1-10 carbon atoms (C₁₋₁₀alkyl). In another embodiment, the alkyl groupemployed contains 1-8 carbon atoms (C₁₋₈ alkyl). In another embodiment,the alkyl group employed contains 1-6 carbon atoms (C₁₋₆alkyl). Inanother embodiment, the alkyl group employed contains 1-5 carbon atoms(C₁₋₅ alkyl). In another embodiment, the alkyl group employed contains1-4 carbon atoms (C₁₋₄alkyl). In another embodiment, the alkyl groupemployed contains 1-3 carbon atoms (C₁₋₃alkyl). In another embodiment,the alkyl group employed contains 1-2 carbon atoms (C₁₋₂alkyl). Examplesof alkyl radicals include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl,iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl,n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like, which maybear one or more substituents. Alkyl group substituents include, but arenot limited to, any of the substituents described herein, that result inthe formation of a stable moiety. The term “alkylene,” as used herein,refers to a biradical derived from an alkyl group, as defined herein, byremoval of two hydrogen atoms. Alkylene groups may be cyclic or acyclic,branched or unbranched, substituted or unsubstituted. Alkylene groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety.

The term “alkenyl,” as used herein, denotes a monovalent group derivedfrom a straight- or branched-chain hydrocarbon moiety having at leastone carbon-carbon double bond by the removal of a single hydrogen atom.In certain embodiments, the alkenyl group employed in the inventioncontains 2-20 carbon atoms (C₂₋₂₀alkenyl). In some embodiments, thealkenyl group employed in the invention contains 2-15 carbon atoms(C₂₋₁₅alkenyl). In another embodiment, the alkenyl group employedcontains 2-10 carbon atoms (C₂₋₁₀alkenyl). In still other embodiments,the alkenyl group contains 2-8 carbon atoms (C₂₋₈alkenyl). In yet otherembodiments, the alkenyl group contains 2-6 carbons (C₂₋₆alkenyl). Inyet other embodiments, the alkenyl group contains 2-5 carbons(C₂₋₅alkenyl). In yet other embodiments, the alkenyl group contains 2-4carbons (C₂₋₄alkenyl). In yet other embodiments, the alkenyl groupcontains 2-3 carbons (C₂₋₃alkenyl). In yet other embodiments, thealkenyl group contains 2 carbons (C₂alkenyl). Alkenyl groups include,for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and thelike, which may bear one or more substituents. Alkenyl groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety. Theterm “alkenylene,” as used herein, refers to a biradical derived from analkenyl group, as defined herein, by removal of two hydrogen atoms.Alkenylene groups may be cyclic or acyclic, branched or unbranched,substituted or unsubstituted. Alkenylene group substituents include, butare not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety.

The term “alkynyl,” as used herein, refers to a monovalent group derivedfrom a straight- or branched-chain hydrocarbon having at least onecarbon-carbon triple bond by the removal of a single hydrogen atom. Incertain embodiments, the alkynyl group employed in the inventioncontains 2-20 carbon atoms (C₂₋₂₀alkynyl). In some embodiments, thealkynyl group employed in the invention contains 2-15 carbon atoms(C₂₋₁₅alkynyl). In another embodiment, the alkynyl group employedcontains 2-10 carbon atoms (C₂₋₁₀alkynyl). In still other embodiments,the alkynyl group contains 2-8 carbon atoms (C₂₋₈alkynyl). In stillother embodiments, the alkynyl group contains 2-6 carbon atoms(C₂₋₆alkynyl). In still other embodiments, the alkynyl group contains2-5 carbon atoms (C₂₋₅alkynyl). In still other embodiments, the alkynylgroup contains 2-4 carbon atoms (C₂₋₄alkynyl). In still otherembodiments, the alkynyl group contains 2-3 carbon atoms (C₂₋₃alkynyl).In still other embodiments, the alkynyl group contains 2 carbon atoms(C₂alkynyl). Representative alkynyl groups include, but are not limitedto, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like, which maybear one or more substituents. Alkynyl group substituents include, butare not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety. The term “alkynylene,” asused herein, refers to a biradical derived from an alkynylene group, asdefined herein, by removal of two hydrogen atoms. Alkynylene groups maybe cyclic or acyclic, branched or unbranched, substituted orunsubstituted. Alkynylene group substituents include, but are notlimited to, any of the substituents described herein, that result in theformation of a stable moiety.

The term “heteroalkyl” refers to an alkyl group, which further includesat least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected fromoxygen, nitrogen, or sulfur within (i.e., inserted between adjacentcarbon atoms of) and/or placed at one or more terminal position(s) ofthe parent chain. In certain embodiments, a heteroalkyl group refers toa saturated group having from 1 to 10 carbon atoms and 1 or moreheteroatoms within the parent chain (“heteroC₁₋₁₀ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 9carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₉ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 8 carbon atoms and 1 or more heteroatomswithin the parent chain (“heteroC₁₋₈ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1or more heteroatoms within the parent chain (“heteroC₁₋₇ alkyl”). Insome embodiments, a heteroalkyl group is a saturated group having 1 to 6carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₆ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms withinthe parent chain (“heteroC₁₋₅ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1or 2 heteroatoms within the parent chain (“heteroC₁₋₄ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 3carbon atoms and 1 heteroatom within the parent chain (“heteroC₁₋₃alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 1 to 2 carbon atoms and 1 heteroatom within the parent chain(“heteroC₁₋₂ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 carbon atom and 1 heteroatom (“heteroC₁alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parentchain (“heteroC₂₋₆ alkyl”). Unless otherwise specified, each instance ofa heteroalkyl group is independently unsubstituted (an “unsubstitutedheteroalkyl”) or substituted (a “substituted heteroalkyl”) with one ormore substituents. In certain embodiments, the heteroalkyl group is anunsubstituted heteroC₁₋₁₀ alkyl. In certain embodiments, the heteroalkylgroup is a substituted heteroC₁₋₁₀ alkyl.

The term “heteroalkenyl” refers to an alkenyl group, which furtherincludes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms)selected from oxygen, nitrogen, or sulfur within (i.e., inserted betweenadjacent carbon atoms of) and/or placed at one or more terminalposition(s) of the parent chain. In certain embodiments, a heteroalkenylgroup refers to a group having from 2 to 10 carbon atoms, at least onedouble bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₁₀ alkenyl”). In some embodiments, a heteroalkenyl group has2 to 9 carbon atoms at least one double bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₉ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 8 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 7 carbonatoms, at least one double bond, and 1 or more heteroatoms within theparent chain (“heteroC₂₋₇ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 6 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbonatoms, at least one double bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 4 carbon atoms, at least one double bond,and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkenyl”).In some embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, atleast one double bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkenyl”). In some embodiments, a heteroalkenyl group has 2to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkenyl”). Unless otherwisespecified, each instance of a heteroalkenyl group is independentlyunsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a“substituted heteroalkenyl”) with one or more substituents. In certainembodiments, the heteroalkenyl group is an unsubstituted heteroC₂₋₁₀alkenyl. In certain embodiments, the heteroalkenyl group is asubstituted heteroC₂₋₁₀ alkenyl.

The term “heteroalkynyl” refers to an alkynyl group, which furtherincludes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms)selected from oxygen, nitrogen, or sulfur within (i.e., inserted betweenadjacent carbon atoms of) and/or placed at one or more terminalposition(s) of the parent chain. In certain embodiments, a heteroalkynylgroup refers to a group having from 2 to 10 carbon atoms, at least onetriple bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₁₀ alkynyl”). In some embodiments, a heteroalkynyl group has2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₉ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 7 carbonatoms, at least one triple bond, and 1 or more heteroatoms within theparent chain (“heteroC₂₋₇ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbonatoms, at least one triple bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond,and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkynyl”).In some embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, atleast one triple bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkynyl”). In some embodiments, a heteroalkynyl group has 2to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkynyl”). Unless otherwisespecified, each instance of a heteroalkynyl group is independentlyunsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a“substituted heteroalkynyl”) with one or more substituents. In certainembodiments, the heteroalkynyl group is an unsubstituted heteroC₂₋₁₀alkynyl. In certain embodiments, the heteroalkynyl group is asubstituted heteroC₂₋₁₀ alkynyl.

The term “carbocyclyl” or “carbocyclic” refers to a radical of anon-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbonatoms (“C₃₋₁₄ carbocyclyl”) and zero heteroatoms in the non-aromaticring system. In some embodiments, a carbocyclyl group has 3 to 10 ringcarbon atoms (“C₃₋₁₀ carbocyclyl”). In some embodiments, a carbocyclylgroup has 3 to 8 ring carbon atoms (“C₃₋₈ carbocyclyl”). In someembodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C₃₋₇carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ringcarbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, a carbocyclylgroup has 4 to 6 ring carbon atoms (“C₄₋₆ carbocyclyl”). In someembodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C₅₋₆carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groupsinclude, without limitation, cyclopropyl (C₃), cyclopropenyl (C₃),cyclobutyl (C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl(C₅), cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and thelike. Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing afused, bridged or spiro ring system such as a bicyclic system (“bicycliccarbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can besaturated or can contain one or more carbon-carbon double or triplebonds. “Carbocyclyl” also includes ring systems wherein the carbocyclylring, as defined above, is fused with one or more aryl or heteroarylgroups wherein the point of attachment is on the carbocyclyl ring, andin such instances, the number of carbons continue to designate thenumber of carbons in the carbocyclic ring system. Unless otherwisespecified, each instance of a carbocyclyl group is independentlyunsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is an unsubstituted C₃₋₁₄carbocyclyl. In certain embodiments, the carbocyclyl group is asubstituted C₃₋₁₄ carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 14 ring carbon atoms (“C₃₋₁₄cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 10 ringcarbon atoms (“C₃₋₁₀ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 8 ring carbon atoms (“C₃₋₈ cycloalkyl”). In someembodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C₃₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ringcarbon atoms (“C₄₋₆ cycloalkyl”). In some embodiments, a cycloalkylgroup has 5 to 6 ring carbon atoms (“C₅₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C₅₋₁₀cycloalkyl”). Examples of C₅₋₆ cycloalkyl groups include cyclopentyl(C₅) and cyclohexyl (C₅). Examples of C₃₋₆ cycloalkyl groups include theaforementioned C₅₋₆ cycloalkyl groups as well as cyclopropyl (C₃) andcyclobutyl (C₄). Examples of C₃₋₈ cycloalkyl groups include theaforementioned C₃₋₆ cycloalkyl groups as well as cycloheptyl (C₇) andcyclooctyl (C₈). Unless otherwise specified, each instance of acycloalkyl group is independently unsubstituted (an “unsubstitutedcycloalkyl”) or substituted (a “substituted cycloalkyl”) with one ormore substituents. In certain embodiments, the cycloalkyl group is anunsubstituted C₃₋₁₄ cycloalkyl. In certain embodiments, the cycloalkylgroup is a substituted C₃₋₁₄ cycloalkyl.

The term “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to14-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or polycyclic (e.g., a fused, bridged or spiro ring system such as abicyclic system (“bicyclic heterocyclyl”) or tricyclic system(“tricyclic heterocyclyl”)), and can be saturated or can contain one ormore carbon-carbon double or triple bonds. Heterocyclyl polycyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.In certain embodiments, the heterocyclyl group is a substituted 3-14membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-8 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl groupis a 5-6 membered non-aromatic ring system having ring carbon atoms and1-4 ring heteroatoms, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azirdinyl, oxiranyl, and thiiranyl.Exemplary 4-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azetidinyl, oxetanyl, and thietanyl.Exemplary 5-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl,and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining 2 heteroatoms include, without limitation, dioxolanyl,oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groupscontaining 3 heteroatoms include, without limitation, triazolinyl,oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclylgroups containing 1 heteroatom include, without limitation, piperidinyl,tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-memberedheterocyclyl groups containing 2 heteroatoms include, withoutlimitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary6-membered heterocyclyl groups containing 2 heteroatoms include, withoutlimitation, triazinanyl. Exemplary 7-membered heterocyclyl groupscontaining 1 heteroatom include, without limitation, azepanyl, oxepanyland thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1heteroatom include, without limitation, azocanyl, oxecanyl andthiocanyl. Exemplary bicyclic heterocyclyl groups include, withoutlimitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl,tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl,octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g.,bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or14π electrons shared in a cyclic array) having 6-14 ring carbon atomsand zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C₁₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systems whereinthe aryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the radical or point of attachment is onthe aryl ring, and in such instances, the number of carbon atomscontinue to designate the number of carbon atoms in the aryl ringsystem. Unless otherwise specified, each instance of an aryl group isindependently unsubstituted (an “unsubstituted aryl”) or substituted (a“substituted aryl”) with one or more substituents. In certainembodiments, the aryl group is an unsubstituted C₆₋₁₄ aryl. In certainembodiments, the aryl group is a substituted C₆₋₁₄ aryl.

The term “heteroaryl” refers to a radical of a 5-14 membered monocyclicor polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system(e.g., having 6, 10, or 14π electrons shared in a cyclic array) havingring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ringsystem, wherein each heteroatom is independently selected from nitrogen,oxygen, and sulfur (“5-14 membered heteroaryl”). In heteroaryl groupsthat contain one or more nitrogen atoms, the point of attachment can bea carbon or nitrogen atom, as valency permits. Heteroaryl polycyclicring systems can include one or more heteroatoms in one or both rings.“Heteroaryl” includes ring systems wherein the heteroaryl ring, asdefined above, is fused with one or more carbocyclyl or heterocyclylgroups wherein the point of attachment is on the heteroaryl ring, and insuch instances, the number of ring members continue to designate thenumber of ring members in the heteroaryl ring system. “Heteroaryl” alsoincludes ring systems wherein the heteroaryl ring, as defined above, isfused with one or more aryl groups wherein the point of attachment iseither on the aryl or heteroaryl ring, and in such instances, the numberof ring members designates the number of ring members in the fusedpolycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groupswherein one ring does not contain a heteroatom (e.g., indolyl,quinolinyl, carbazolyl, and the like) the point of attachment can be oneither ring, i.e., either the ring bearing a heteroatom (e.g.,2-indolyl) or the ring that does not contain a heteroatom (e.g.,5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently unsubstituted (an “unsubstituted heteroaryl”) orsubstituted (a “substituted heteroaryl”) with one or more substituents.In certain embodiments, the heteroaryl group is an unsubstituted 5-14membered heteroaryl. In certain embodiments, the heteroaryl group is asubstituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include,without limitation, pyrrolyl, furanyl, and thiophenyl. Exemplary5-membered heteroaryl groups containing 2 heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing 3heteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4heteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing 1 heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, andpyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4heteroatoms include, without limitation, triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing 1heteroatom include, without limitation, azepinyl, oxepinyl, andthiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, withoutlimitation, indolyl, isoindolyl, indazolyl, benzotriazolyl,benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl,benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl,benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, andpurinyl. Exemplary 6,6-bicyclic heteroaryl groups include, withoutlimitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplarytricyclic heteroaryl groups include, without limitation,phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl,phenoxazinyl and phenazinyl.

The term “unsaturated” or “partially unsaturated” refers to a moietythat includes at least one double or triple bond.

Affixing the suffix “-ene” to a group indicates the group is a divalentmoiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene isthe divalent moiety of alkenyl, alkynylene is the divalent moiety ofalkynyl, heteroalkylene is the divalent moiety of heteroalkyl,heteroalkenylene is the divalent moiety of heteroalkenyl,heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclyleneis the divalent moiety of carbocyclyl, heterocyclylene is the divalentmoiety of heterocyclyl, arylene is the divalent moiety of aryl, andheteroarylene is the divalent moiety of heteroaryl.

A group is optionally substituted unless expressly provided otherwise.In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl groups are optionally substituted. “Optionally substituted”refers to a group which may be substituted or unsubstituted (e.g.,“substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted”alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” heteroalkyl, “substituted” or “unsubstituted”heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl,“substituted” or “unsubstituted” carbocyclyl, “substituted” or“unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or“substituted” or “unsubstituted” heteroaryl group). In general, the term“substituted” means that at least one hydrogen present on a group isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound, e.g., a compound which doesnot spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction. Unless otherwise indicated,a “substituted” group has a substituent at one or more substitutablepositions of the group, and when more than one position in any givenstructure is substituted, the substituent is either the same ordifferent at each position. The term “substituted” is contemplated toinclude substitution with all permissible substituents of organiccompounds, and includes any of the substituents described herein thatresults in the formation of a stable compound. The present inventioncontemplates any and all such combinations in order to arrive at astable compound. For purposes of this invention, heteroatoms such asnitrogen may have hydrogen substituents and/or any suitable substituentas described herein which satisfy the valencies of the heteroatoms andresults in the formation of a stable moiety. The invention is notintended to be limited in any manner by the exemplary substituentsdescribed herein.

The term “halo” or “halogen” refers to fluorine (fluoro, —F), chlorine(chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

The term “amino” refers to the group —NH₂. The term “substituted amino,”by extension, refers to a monosubstituted amino, a disubstituted amino,or a trisubstituted amino. In certain embodiments, the “substitutedamino” is a monosubstituted amino or a disubstituted amino group.

The term “monosubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith one hydrogen and one group other than hydrogen, and includes groupsselected from —NH(R^(bb)), —NHC(═O)R^(aa), —NHCO₂R^(aa),—NHC(═O)N(R^(bb))₂, —NHC(═NR^(bb))N(R^(bb))₂, —NHSO₂R^(aa),—NHP(═O)(OR^(cc))₂, and —NHP(═O)(NR^(bb))₂, wherein R^(aa), R^(bb) andR^(cc) are as defined herein, and wherein R^(bb) of the group—NH(R^(bb)) is not hydrogen.

The term “disubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith two groups other than hydrogen, and includes groups selected from—N(R^(bb))₂, —NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa),—NR^(bb)C(═O)N(R^(bb))₂, —NR^(bb)C(═NR^(bb))N(R^(bb))₂,—NR_(bb)SO₂R^(aa), —NR^(bb)P(═O)(OR^(cc))₂, and —NR^(bb)P(═O)(NR^(bb))₂,wherein R^(aa), R^(bb), and R^(cc) are as defined herein, with theproviso that the nitrogen atom directly attached to the parent moleculeis not substituted with hydrogen.

The term “trisubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith three groups, and includes groups selected from —N(R^(bb))₃ and—N(R^(bb))₃ ⁺X⁻, wherein R^(bb) and X⁻ are as defined herein.

In certain embodiments, the substituent present on the nitrogen atom isan nitrogen protecting group (also referred to herein as an “aminoprotecting group”). Nitrogen protecting groups include, but are notlimited to, —OH, —OR^(aa), —N(R)₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂,—CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R and R^(dd) are as definedherein. Nitrogen protecting groups are well known in the art and includethose described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999,incorporated herein by reference.

For example, nitrogen protecting groups such as amide groups (e.g.,—C(═O)R^(aa)) include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g.,—C(═O)OR^(aa)) include, but are not limited to, methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc),vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallylcarbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate(Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g.,—S(═O)₂R^(aa)) include, but are not limited to, p-toluenesulfonamide(Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide(Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl] methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to herein as an “hydroxylprotecting group”). Oxygen protecting groups include, but are notlimited to, —R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa),—CO₂R^(aa), —C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethylcarbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate(Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc),isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate(BOC or Boc), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzylcarbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate,p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-naphthylcarbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate,4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, o-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

In certain embodiments, the substituent present on an sulfur atom is asulfur protecting group (also referred to as a “thiol protectinggroup”). Sulfur protecting groups include, but are not limited to,—R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Sulfur protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

The terms “aminooxy,” or “aminooxy group,” are used interchangeablyherein and refer to functional groups having the general formula:

herein R³¹ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, optionally substitutedheteroaryl, or optionally substituted heterocyclyl. In some embodiments,R³¹ is an amino acid, wherein the point of attachment for the oxygen ison the side chain of the amino acid. In certain embodiments, the aminoacid is within a polypeptide.

The term “tautomers” or “tautomeric” refers to two or moreinterconvertable compounds resulting from at least one formal migrationof a hydrogen atom and at least one change in valency (e.g., a singlebond to a double bond, a triple bond to a single bond, or vice versa).The exact ratio of the tautomers depends on several factors, includingtemperature, solvent, and pH. Tautomerizations (i.e., the reactionproviding a tautomeric pair) may catalyzed by acid or base. Exemplarytautomerizations include keto-to-enol, amide-to-imide, lactam-to-lactim,enamine-to-imine, and enamine-to-(a different enamine) tautomerizations.

It is also to be understood that compounds that have the same molecularformula but differ in the nature or sequence of bonding of their atomsor the arrangement of their atoms in space are termed “isomers”. Isomersthat differ in the arrangement of their atoms in space are termed“stereoisomers”.

Stereoisomers that are not mirror images of one another are termed“diastereomers” and those that are non-superimposable mirror images ofeach other are termed “enantiomers”. When a compound has an asymmetriccenter, for example, it is bonded to four different groups, a pair ofenantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is described by theR- and S-sequencing rules of Cahn and Prelog, or by the manner in whichthe molecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e., as (+) or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a “racemic mixture”.

The terms “carbonyl,” or “carbonyl group,” are used interchangeablyherein and refer to functional groups composed of a carbon atomdouble-bonded to any oxygen atom. Carbonyls have the general formula:

wherein each of R³² and R³³ independently represents hydroxyl,optionally substituted amino, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl,optionally substituted heteroaryl, or optionally substitutedheterocyclyl. Examples of carbonyls include, but are not limited to,aldehydes, ketones, carboxylic acids, esters, amides, enones, acylhalides, acid anhydrides, and imides. In some embodiments, acarbonyl-containing compound refers to a compound having an aldehydegroup, or a compound capable of forming an aldehyde group throughisomerization. For example, in some embodiments, certain sugars (e.g.,reducing sugars) such as glucose, form aldehydes through isomerization.A sugar is classified as a reducing sugar if it has an open-chain formwith an aldehyde group or a free hemiacetal group. Monosaccharides whichcontain an aldehyde group are known as aldoses, and those with a ketonegroup are known as ketoses. The aldehyde can be oxidized via a redoxreaction in which another compound is reduced. Thus, a reducing sugar isone that is capable of reducing certain chemicals. Sugars with ketonegroups in their open chain form are capable of isomerizing via a seriesof tautomeric shifts to produce an aldehyde group in solution.Therefore, ketone-bearing sugars like fructose are considered reducingsugars but it is the isomer containing an aldehyde group which isreducing since ketones cannot be oxidized without decomposition of thesugar. This type of isomerization is catalyzed by the base present insolutions which test for the presence of aldehydes.

The term “hydrazide,” as used herein, refers to functional groups havingthe general formula:

wherein R³⁴ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, optionally substitutedheteroaryl, or optionally substituted heterocyclyl. In some embodiments,R³⁴ is an amino acid, wherein the point of attachment for the oxygen ison the side chain of the amino acid. In certain embodiments, the aminoacid is within a polypeptide.

The term “hydrazone,” as used herein, refers to compound having thegeneral formula:

wherein each of R³⁵, R³⁶, and R³⁷ is independently optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, optionally substituted heteroaryl, oroptionally substituted heterocyclyl. The term “hydrazone linkage,” asused herein, refers to the formula:

Hydrzones can be prepared from, for example, joining of a compoundcomprising a hydrazide group and a compound comprising a carbonyl.

The term “acyl,” as used herein, is a subset of a substituted alkylgroup, and refers to a group having the general formula —C(═O)R^(A),—C(═O)OR^(A), —C(═O)—O—C(═O)R^(A), —C(═O)SR^(A), or —C(═O)N(R^(A))₂,wherein each instance of R^(A) is independently hydrogen, optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, optionally substituted heteroaryl, oroptionally substituted heterocyclyl. Exemplary acyl groups includealdehydes (—CHO), carboxylic acids (—CO₂H), ketones, acyl halides,esters, amides, imines, carbonates, carbamates, and ureas. Acylsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety.

The term “azide” or “azido,” as used herein, refers to a group of theformula (—N₃).

Radiolabled Proteins

Aspects of the disclosure provide antibodies, or fragments thereof,(e.g., single domain antibodies) comprising a radiolabel and/or ahydrophilic polymer. It should be appreciated that any of the linkagesprovided herein may be used to link proteins (e.g., antibodies) to aradiolabel and/or a hydrophilic polymer. It should also be appreciatedthat agents, such as the chelating moieties and radionuclides providedbelow may be substituted with another agents, such as any of thechelating moieties and radionuclides provided herein

Another aspect of the present invention provides radiolabeled proteins.The radiolabeled proteins can be prepared from modified proteins ofFormula (I).

In certain embodiments, provided herein is a modified protein of Formula(I):

wherein

L¹ is a linker comprising at least four amino acids formed by enzymaticconjugation between two enzyme recognition sequences; and

R¹ comprises a reactive group capable of undergoing a click chemistryreaction.

As generally defined herein, L¹ is a linker formed by enzymaticconjugation between two enzyme recognition sequences. In certainembodiments, L¹ comprises at least four amino acids. In certainembodiments, L¹ comprises at least five amino acids. In certainembodiments, L¹ comprises at least six amino acids. In certainembodiments, L¹ comprises at least seven amino acids. In certainembodiments, L¹ is a linker formed by sortase-mediated transpeptidationof two sortase recognition sequences. In certain embodiments, L¹ is-LPXTGGGK-, -LPXTGGG-, -NPXTGGGK-, NPXTGGG-, -LPXTAAA-, -NPXTAAA-,-LPXTGGGGG-, or -LPGAG-, wherein each instance of X is independently anamino acid. In certain embodiments, X is E. In certain embodiments, X isQ. In certain embodiments, X is K.

In certain embodiments, the modified protein is formed by enzymaticconjugation of

and a compound of Formula (a): B—R¹ (a), wherein each of A and B isindependently an enzyme recognition sequence. In certain embodiments,the modified protein is formed by sortase-mediated transpeptidation of

and the compound of Formula (a): B—R¹ (a), wherein A comprises aC-terminal sortase recognition sequence, and B comprises a N-terminalsortase recognition sequence; or A comprises a N-terminal sortaserecognition sequence, and B comprises a C-terminal sortase recognitionsequence.

In certain embodiments, A comprises LPXTX or NPXTX, and B comprises anoligoglycine or an oligoalanine sequence; wherein each instance of X isindependently an amino acid. In certain embodiments, B comprises LPXTXor NPXTX, and A comprises an oligoglycine or an oligoalanine sequence;wherein each instance of X is independently an amino acid. In certainembodiments, A is LPETG, LPETA, NPQTN, or NPKTG, and B is GGG or AAA. Incertain embodiments, A comprises an oligoglycine or an oligoalaninesequence, and B comprises LPXTX or NPXTX, wherein each instance of X isindependently an amino acid. In certain embodiments, B is LPETG, LPETA,NPQTN, or NPKTG, and A is GGG or AAA.

As used herein, the enzyme recognition sequence is an amino acidsequence recognized by a transamidase enzyme. In certain embodiments,the transamidase recognition sequence is a sortase recognition sequenceor a sortase recognition motif. In certain embodiments, the sortase issortase A (SrtA). In certain embodiments, the sortase is sortase B(SrtB).

As generally defined herein, R¹ is a reactive group capable ofundergoing a click chemistry reaction.

Click chemistry is a chemical approach introduced by Sharpless in 2001and describes chemistry tailored to generate substances quickly andreliably by joining small units together. See, e.g., Kolb, Finn andSharpless Angewandte Chemie International Edition (2001) 40: 2004-2021;Evans, Australian Journal of Chemistry (2007) 60: 384-395). Exemplarycoupling reactions (some of which may be classified as “Clickchemistry”) include, but are not limited to, formation of esters,thioesters, amides (e.g., such as peptide coupling) from activated acidsor acyl halides; nucleophilic displacement reactions (e.g., such asnucleophilic displacement of a halide or ring opening of strained ringsystems); azide-alkyne Huisgon cycloaddition; thiol-yne addition; imineformation; Michael additions (e.g., maleimide addition); Diels-Alderreaction and inverse electron demand Diels-Alder reaction; and [4+1]cycloadditions (e.g. between isonitriles (isocyanides) and tetrazines).In certain embodiments, the click chemistry reaction is a Diels-Alderreaction. It is to be understood that the click chemistry reaction maybe followed by additional one or more chemical transformations. Incertain embodiments, the click chemistry reaction is a Diels-Alderreaction followed by an retro-Diels-Alder reaction.

In certain embodiments, R¹ is a reactive group capable of undergoing a[3+2]cycloaddition. In certain embodiments, R¹ comprises adipolarophile. In certain embodiments, R¹ comprises an alkynyl group. Incertain embodiments, R¹ comprises a 1,3-dipole. In certain embodiments,R¹ comprises an azido. In certain embodiments, R¹ is a reactive groupcapable of undergoing a Diels-Alder cycloaddition. In certainembodiments, R¹ comprises a conjugated diene. In certain embodiments, R¹comprises a tetrazine or a quadricyclane. In certain embodiments, R¹comprises a tetrazine. In certain embodiments, R¹ comprises anunsubstituted tetrazine. In certain embodiments, R¹ comprises asubstituted tetrazine.

In certain embodiments, R¹ is of Formula (i):

wherein

R^(t) is hydrogen, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted aryl, optionallysubstituted heteroaryl, or optionally substituted heterocyclyl; and

R^(s) is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene.

As generally defined herein, R^(t) is hydrogen, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted aryl, optionally substituted heteroaryl, or optionallysubstituted heterocyclyl. In certain embodiments, R^(t) is hydrogen. Incertain embodiments, R^(t) is optionally substituted aliphatic. Incertain embodiments, R^(t) is optionally substituted C₁₋₆ alkyl. Incertain embodiments, R^(t) is unsubstituted C₁₋₆ alkyl. In certainembodiments, R^(t) is methyl or ethyl. In certain embodiments, R^(t) issubstituted C₁₋₆ alkyl. In certain embodiments, R^(t) is optionallysubstituted aryl. In certain embodiments, R^(t) is optionallysubstituted phenyl. In certain embodiments, R^(t) is optionallysubstituted heteroaryl. In certain embodiments, R^(t) is optionallysubstituted pyridine.

As generally defined herein, R^(s) is a bond, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted arylene, optionally substituted heteroarylene, or optionallysubstituted heterocyclylene. In certain embodiments, R^(s) is a bond. Incertain embodiments, R^(s) is optionally substituted aliphatic. Incertain embodiments, R^(s) is optionally substituted C₁₋₆ alkyl. Incertain embodiments, R^(s) is unsubstituted C₁₋₆ alkyl. In certainembodiments, R^(s) is methyl or ethyl. In certain embodiments, R^(s) issubstituted C₁₋₆ alkyl. In certain embodiments, R^(s) is optionallysubstituted heteroaliphatic. In certain embodiments, R^(s) is optionallysubstituted arylene. In certain embodiments, R^(s) is optionallysubstituted phenyl. In certain embodiments, R^(s) is optionallysubstituted heteroarylene. In certain embodiments, R^(s) is optionallysubstituted pyridine.

In certain embodiments, R¹ comprises a dienophile. In certainembodiments, R¹ comprises an optionally substituted alkene. In certainembodiments, R¹ comprises a cyclooctene. In certain embodiments, R¹comprises a substituted cyclooctene of the formula:

wherein R^(A1) is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene. R¹comprises a substituted cyclooctene of the formula:

In certain embodiments, R¹ comprises a trans-cyclooctene. In certainembodiments, R¹ comprises a substituted trans-cyclooctene of the formula

wherein R^(A1) is as defined herein. In certain embodiments, R¹comprises a substituted trans-cyclooctene of the formula

wherein R^(A1) is as defined herein. In certain embodiments, R¹comprises an unsubstituted trans-cyclooctene.

Another aspect of the invention provides a radioactive protein ofFormula (II)

wherein

L¹ is a linker comprising at least four amino acids formed by enzymaticconjugation between two enzyme recognition sequences; and

L² is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted arylene, optionally substitutedheteroarylene, or optionally substituted heterocyclylene.

In certain embodiments, the linker L² is formed by a click chemistryreaction. In certain embodiments, the linker L² is formed by a [3+2]cycloaddition. In certain embodiments, the linker L² is formed by aDiels-Alder cycloaddition. In certain embodiments, the linker L² isformed by a Diels-Alder cycloaddition followed by one or more chemicaltransformations. In certain embodiments, the linker L² is formed by aDiels-Alder cycloaddition followed by retro-Diels-Alder reaction.

As generally defined herein, L² is optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted arylene,optionally substituted heteroarylene, or optionally substitutedheterocyclylene. In certain embodiments, L² is optionally substitutedaliphatic. In certain embodiments, L² is optionally substitutedheteroaliphatic. In certain embodiments, L² is optionally substitutedarylene. In certain embodiments, L² is optionally substitutedcycloalkylene. In certain embodiments, L² is optionally substitutedheteroarylene.

In certain embodiments, the radioactive protein of Formula (II) isformed by a click chemistry reaction of the modified protein of Formula(I) and a compound of Formula (b): ¹⁸F—R² (b), wherein R² is a reactivegroup capable of undergoing the click chemistry reaction.

As generally defined herein, R² is a reactive group capable ofundergoing a click chemistry reaction. In certain embodiments, R² is areactive unsaturated group capable of undergoing a [3+2] cycloaddition.In certain embodiments, R² comprises a dipolarophile. In certainembodiments, R² comprises an alkynyl group. In certain embodiments, R²comprises a 1,3-dipole. In certain embodiments, R² comprises an azido.In certain embodiments, R² is a reactive group capable of undergoing aDiels-Alder cycloaddition. In certain embodiments, R² comprises aconjugated diene. In certain embodiments, R² comprises a tetrazine or aquadricyclane. In certain embodiments, R² comprises a tetrazine. Incertain embodiments, R² comprises an unsubstituted tetrazine. In certainembodiments, R² comprises a substituted tetrazine.

In certain embodiments, R² is of Formula (i):

wherein

R^(t) is hydrogen, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted aryl, optionallysubstituted heteroaryl, or optionally substituted heterocyclyl; and

R^(s) is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene.

In certain embodiments, R² comprises a dienophile. In certainembodiments, R² comprises an optionally substituted alkene. In certainembodiments, R² comprises a cyclooctene. In certain embodiments, R²comprises a trans-cyclooctene. In certain embodiments, R² comprises asubstituted trans-cyclooctene. In certain embodiments, R² comprises anunsubstituted trans-cyclooctene.

In certain embodiments, a compound of Formula (b): ¹⁸F—R² (b), is ofFormula (b-1):

wherein R^(A2) is optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted aryl, optionallysubstituted heteroaryl, or optionally substituted heterocyclyl; andR^(A2) comprises ¹⁸F. In certain embodiments, R^(A2) is optionallysubstituted aliphatic. In certain embodiments, R^(A2) is optionallysubstituted heteroaliphatic. In certain embodiments, R^(A2) is—O—C₁₋₆alkylene, wherein the C₁₋₆alkylene comprises ¹⁸F.

In certain embodiments, a compound of Formula (b) is of Formula (b-1-a):

wherein h is an integer of 1 to 5, inclusive.

Compounds of Formula b-1-a) can be prepared from a compound of Formula(b-1-a-i):

with a proper nucleophile comprising ¹⁸F, wherein h is as definedherein, LG is a leaving group. In certain embodiments, the compounds ofFormula (b-1) can be prepared according to Scheme S6 or Scheme S6-a.

The term “leaving group” is given its ordinary meaning in the art ofsynthetic organic chemistry and refers to an atom or a group capable ofbeing displaced by a nucleophile. Examples of suitable leaving groupsinclude, but are not limited to, halogen (such as F, Cl, Br, or I(iodine)), alkoxycarbonyloxy, aryloxycarbonyloxy, alkanesulfonyloxy,arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy), arylcarbonyloxy,aryloxy, methoxy, N,O-dimethylhydroxylamino, pixyl, and haloformates. Insome cases, the leaving group is a sulfonic acid ester, such astoluenesulfonate (tosylate, —OTs), methanesulfonate (mesylate, —OMs),p-bromobenzenesulfonyloxy (brosylate, —OBs), ortrifluoromethanesulfonate (triflate, —OTf). In some cases, the leavinggroup is a brosylate, such as p-bromobenzenesulfonyloxy. In some cases,the leaving group is a nosylate, such as 2-nitrobenzenesulfonyloxy. Insome embodiments, the leaving group is a sulfonate-containing group. Insome embodiments, the leaving group is a tosylate group. The leavinggroup may also be a phosphineoxide (e.g., formed during a Mitsunobureaction) or an internal leaving group such as an epoxide or cyclicsulfate. Other non-limiting examples of leaving groups are water,ammonia, alcohols, ether moieties, thioether moieties, zinc halides,magnesium moieties, diazonium salts, and copper moieties. In certainembodiments, LG is -OTs.

In certain embodiments, a compound of Formula (b) is of Formula (b-1-b):

wherein L⁴ and Y are as defined herein.

In certain embodiments, a compound of Formula (b) is of Formula (b-1-c):

wherein L⁴, M, L^(a), L^(b), and L^(c) are as defined herein, and ---indicates a coordination bond or absent, as valency permits. In certainembodiments, ---- is a single coordination bond. In certain embodiments,--- is absent.

In certain embodiments, a compound of Formula (b) is of the formula:

In certain embodiments, a compound of Formula (b): ¹⁸F—R² (b), is ofFormula (b-2):

wherein R^(s) and R^(t) are as defined herein.

In certain embodiments, a compound of Formula (b) is of Formula (b-2-a):

wherein R^(t), R^(P), and p are as defined herein.

In certain embodiments, a compound of Formula (b) is of Formula(b-2-a1):

wherein R^(t), R^(N1), L³, R^(q1), and q1 are as defined herein; andR^(G) is an optionally substituted carbohydrate group; provided thatR^(G) comprises ¹⁸F.

A “carbohydrate group” or a “carbohydrate” refers to a monosaccharide ora polysaccharide (e.g., a disaccharide or oligosaccharide). Exemplarymonosaccharides include, but are not limited to, natural sugars, such asallose, altrose, glucose, mannose, gulose, idose, galactose, talose,ribose, arabinose, xylose, and lyxose. Disaccharides are two joinedmonosaccharides. Exemplary disaccharides include, but are not limitedto, sucrose, maltose, cellobiose, and lactose. Typically, anoligosaccharide includes between three and ten monosaccharide units(e.g., raffinose, stachyose). The carbohydrate group may be a naturalsugar or a modified sugar. Exemplary modified sugars include, but arenot limited to, sugars where the hydroxyl group is replaced with anamino group and/or alkyl group (e.g., such as desosamine),2′-deoxyribose wherein a hydroxyl group is removed, 2′-fluororibosewherein a hydroxyl group is replace with a fluorine, orN-acetylglucosamine, or a nitrogen-containing form of glucose (e.g.,2′-fluororibose, deoxyribose, and hexose), and the like. Variouscarbohydrates are further described below and herein. Carbohydrates mayexist in many different forms, for example, conformers, cyclic forms,acyclic forms, stereoisomers, tautomers, anomers, and isomers. Incertain embodiments, R^(G) is an optionally substituted glucose.

In certain embodiments, a compound of Formula (b) is of Formula(b-2-a2):

wherein R^(t), R^(N1) are as defined herein; and R^(G1) is an optionallysubstituted carbohydrate group or a fragment thereof; provided thatR^(G1) comprises ¹⁸F.

The oxime compounds of Formula (b-2-a2)

can be prepared from optionally substituted tetrazine-aminooxy and aradiolabeled optionally substituted aldehyde of the formula R^(as)—CHO,wherein R^(as) is optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted aryl, optionallysubstituted heteroaryl, or optionally substituted heterocyclyl (SchemeSi). In certain embodiments, R^(as) is an optionally substitutedcarbohydrate group or a fragment thereof. In certain embodiments, R^(as)is an optionally substituted glucose or a fragment thereof. In certainembodiments, the reaction is carried out in the presence of a catalyst.In certain embodiments, the catalyst is m-phenylenediamine,p-phenylenediamine, or p-anisidine. In certain embodiments, the catalystis m-phenylenediamine. In certain embodiments, the molar ratio of theoptionally substituted tetrazine-aminooxy to the catalyst is from about10:1 to 1:10. In certain embodiments, the molar ratio of the optionallysubstituted tetrazine-aminooxy to the catalyst is from about 1:1 to 1:8.In certain embodiments, the molar ratio of the optionally substitutedtetrazine-aminooxy to the catalyst is from about 1:1 to 1:6. In certainembodiments, the molar ratio of the optionally substitutedtetrazine-aminooxy to the catalyst is from about 1:2 to 1:4. In certainembodiments, the molar ratio of the optionally substitutedtetrazine-aminooxy to the catalyst is about 1:4.

In certain embodiments, R^(as) is an optionally substituted carbohydrategroup or a fragment thereof, provided R^(as) comprises ¹⁸F. In certainembodiments, R^(as) is an optionally substituted glucose or a fragmentthereof. In certain embodiments, R^(as) is ¹⁸F-FDG of a fragmentthereof.

As provided in Scheme S1, the resulting oxime product can be easilypurified from the reaction mixture to the change in hydrophilicity.

In certain embodiments of Scheme S1, the excess of tetrazine-aminooxycan be captured by reacting with another water soluble carbohydrate. Incertain embodiments, the water soluble carbohydrate is glucosamine6-sulfate.

In certain embodiments, a compound of Formula (b) is of Formula(b-2-a2):

wherein R^(t), R^(N1), L³, R^(s1), R^(s2), R^(s3), and R^(s4) are asdefined herein.

In certain embodiments, a compound of Formula (b) is of Formula(b-2-a3):

wherein R^(t), R^(N1), L³, R^(s1), R^(s2), R^(s3), and R^(s4) are asdefined herein.

In certain embodiments, a compound of Formula (b) is of Formula (b-2-b):

wherein R^(t), R^(N1), L³, R^(g1), and q1 are as defined herein.

In certain embodiments, a compound of Formula (b) is of Formula(b-2-b1):

In certain embodiments, a compound of Formula (b) is of Formula (b-3):

wherein R^(t), L⁴, and Y are as defined herein.

In certain embodiments, a compound of Formula (b) is of Formula (b-3-a):

wherein R^(t), L⁴, L^(a), L^(b), and L^(c) are as defined herein.

In certain embodiments, a compound of Formula (b) is of the followingformula:

In certain embodiments, the linker L² is of Formula (ii):

wherein

R^(t) is hydrogen, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted aryl, optionallysubstituted heteroaryl, or optionally substituted heterocyclyl;

R^(s) is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene;

each instance of R^(c1) is hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl,optionally substituted heteroaryl, or optionally substitutedheterocyclyl; or optionally two R^(c1) taken with the intervening atomsto form an optionally substituted carbocyclyl or optionally substitutedheterocyclyl ring;

R^(c2) is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene;

m is 0, 1, 2, 3, 4, 5, 6, 7, or 8, as valency permits;

a indicates point of attachment to L¹; and

b indicates point of attachment to ¹⁸F.

In certain embodiments, m is 0. In certain embodiments, m is 1. Incertain embodiments, m is 2. In certain embodiments, m is 3. In certainembodiments, m is 4.

As generally defined herein, each instance of R^(c1) is independentlyhydrogen, optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, optionally substitutedheteroaryl, or optionally substituted heterocyclyl. In certainembodiments, R^(c1) is hydrogen. In certain embodiments, R^(c1) isoptionally substituted aliphatic. In certain embodiments, R^(c1) isoptionally substituted alkyl. In certain embodiments, R^(c1) isoptionally substituted heteroaliphatic. In certain embodiments, twoR^(c1) taken with the intervening atoms to form an optionallysubstituted carbocyclyl. In certain embodiments, two R^(c1) taken withthe intervening atoms to form an optionally substituted cyclopropyl.

As generally defined herein, R^(c2) is a bond, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted arylene, optionally substituted heteroarylene, or optionallysubstituted heterocyclylene. In certain embodiments, R^(c2) is a bond.In certain embodiments, R^(c2) is optionally substituted aliphatic. Incertain embodiments, R^(c2) is optionally substituted alkyl. In certainembodiments, R^(c2) is optionally substituted heteroaliphatic. Incertain embodiments, R^(c2) is optionally substituted alkoxy. In certainembodiments, R^(c2) is an optionally substituted amino group.

In certain embodiments, the linker L² is of Formula (ii-a):

wherein n is an integer between 1 and 8, inclusive.

In certain embodiments, n is 1. In certain embodiments, n is 2. Incertain embodiments, n is 3. In certain embodiments, n is 4. In certainembodiments, n is 5. In certain embodiments, n is 6. In certainembodiments, n is 7. In certain embodiments, n is 8.

In certain embodiments, the linker L² is of Formula (ii-b):

wherein n is an integer between 1 and 8, inclusive.

In certain embodiments, the linker L² is of Formula (iii):

wherein

R^(t) is hydrogen, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted aryl, optionallysubstituted heteroaryl, or optionally substituted heterocyclyl;

R^(s) is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene;each instance of R^(c1) is hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl,optionally substituted heteroaryl, or optionally substitutedheterocyclyl;

R^(c2) is a bond, optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted arylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene;

m is 0, 1, 2, 3, 4, 5, 6, 7, or 8, as valency permits;

a indicates point of attachment to L¹; and

b indicates point of attachment to ¹⁸F.

In certain embodiments, the linker L² is of Formula (iii-a):

In certain embodiments, wherein -L²-F⁸ is of Formula (iii-b):

wherein

each instance of R^(p) is independently hydrogen, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted heterocyclyl, hydroxyl, or optionally substituted amino;provided at least one R^(p) is not hydrogen and comprises F¹⁸; and

p is 1, 2, 3, 4, or 5.

In certain embodiments, p is 1. In certain embodiments, p is 2. Incertain embodiments, p is 3. In certain embodiments, p is 4. In certainembodiments, p is 3. In certain embodiments, p is 5.

As generally defined herein, R^(p) is hydrogen, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted heterocyclyl, hydroxyl, or optionally substituted amino;wherein at least one R^(p) is not hydrogen and comprises F¹⁸. In certainembodiments, R^(p) is hydrogen. In certain embodiments, R^(p) isoptionally substituted aliphatic. In certain embodiments, R^(p) isoptionally substituted C₁₋₆ alkyl. In certain embodiments, R^(p) isunsubstituted C₁₋₆ alkyl. In certain embodiments, R^(p) is methyl orethyl. In certain embodiments, R^(p) is substituted C₁₋₆ alkyl. Incertain embodiments, R^(p) is optionally substituted aryl. In certainembodiments, R^(p) is optionally substituted phenyl. In certainembodiments, R^(p) is optionally substituted heteroaryl. In certainembodiments, R^(p) is optionally substituted pyridine. In certainembodiments, least one R^(p) is not hydrogen and comprises F⁸.

In certain embodiments, wherein -L²-F¹⁸ is of Formula (iii-b1):

wherein

L³ is a bond, optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted arylene, optionally substitutedheteroarylene, or optionally substituted heterocyclylene;

R^(N1) is independently hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl, oroptionally substituted heteroaryl, or a nitrogen protecting group; and

each of R^(s1), R^(s2), R^(s3), and R^(s4) is independently hydrogen,optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, or optionally substitutedheteroaryl, or an oxygen protecting group.

As generally defined herein, L³ is a bond, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted arylene, optionally substituted heteroarylene, or optionallysubstituted heterocyclylene. In certain embodiments, L³ is a bond. Incertain embodiments, L³ is optionally substituted aliphatic. In certainembodiments, L³ is optionally substituted heteroaliphatic. In certainembodiments, L³ comprises an oxime moiety. In certain embodiments, L³ isof the formula

wherein c indicates the point of attachment to —N—R^(N1)—; d indicatesthe point of attachment to —CH¹⁸F—; and u is 1, 2, 3, 4, or 5. Incertain embodiments, L³ is C═O.

In certain embodiments, R^(N1) is independently hydrogen. In certainembodiments, R^(N1) is optionally substituted aliphatic. In certainembodiments, R^(N1) is optionally substituted alkyl. In certainembodiments, R^(N1) is an amino protectin group.

In certain embodiments, R^(s1) is independently hydrogen. In certainembodiments, R^(s1) is optionally substituted aliphatic. In certainembodiments, R^(s1) is optionally substituted alkyl. In certainembodiments, R^(s1) is an oxygen protectin group. In certainembodiments, R^(s1) is acyl (e.g. acetyl).

In certain embodiments, R^(s2) is independently hydrogen. In certainembodiments, R^(s2) is optionally substituted aliphatic. In certainembodiments, R^(s2) is optionally substituted alkyl. In certainembodiments, R^(s2) is an oxygen protectin group. In certainembodiments, R^(s2) is acyl (e.g. acetyl).

In certain embodiments, R^(s3) is independently hydrogen. In certainembodiments, R^(s3) is optionally substituted aliphatic. In certainembodiments, R^(s3) is optionally substituted alkyl. In certainembodiments, R^(s3) is an oxygen protectin group. In certainembodiments, R^(s3) is acyl (e.g. acetyl).

In certain embodiments, R^(s4) is independently hydrogen. In certainembodiments, R^(s4) is optionally substituted aliphatic. In certainembodiments, R^(s4) is optionally substituted alkyl. In certainembodiments, R^(s4) is an oxygen protectin group. In certainembodiments, R^(s4) is acyl (e.g. acetyl).

In certain embodiments, R^(s1), R^(s2), R^(s3), and R^(s4) are allhydrogen.

In certain embodiments, R^(N1), R^(s1), R^(s2), R^(s3), and R^(s4) areall hydrogen.

In certain embodiments, R^(N1), R^(s1), R^(s2), R^(s3), and R^(s4) areall hydrogen; and R^(t) is optionally substituted aliphatic. In certainembodiments, R^(N1), R^(s1), R^(s2), R^(s3), and R^(s4) are allhydrogen; and R^(t) is optionally substituted C₁₋₆ alkyl. In certainembodiments, R^(N1), R^(s1), R^(s2), R^(s3) and R^(s4) are all hydrogen;and R^(t) is methyl or ethyl.

In certain embodiments, wherein -L²-F¹⁸ is of the formula:

In certain embodiments, wherein -L²-F⁸ is of Formula (iii-b2):

wherein

L³ is a bond, optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted arylene, optionally substitutedheteroarylene, or optionally substituted heterocyclylene;

R^(N1) is independently hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl, oroptionally substituted heteroaryl, or a nitrogen protecting group;

each instance of R^(q1) is independently hydrogen, optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocyclyl, hydroxyl, or optionally substitutedamino; and

q1 is 0, 1, 2, 3, or 4.

In certain embodiments, R^(t) is optionally substituted aliphatic andR^(q1) is hydrogen. In certain embodiments, R^(t) is optionallysubstituted C₁₋₆ alkyl; L³ is

u is 1; and R^(q1) is hydrogen.

In certain embodiments, -L²-F¹⁸ is of the formula:

In certain embodiments, -L²-F¹⁸ is of Formula (iii-b3):

wherein

R^(N1) is independently hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl, oroptionally substituted heteroaryl, or a nitrogen protecting group; and

each instance of R² is independently hydrogen, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted aryl, optionally substituted carbocyclyl, optionallysubstituted heteroaryl, optionally substituted heterocyclyl, hydroxyl,or optionally substituted amino; and

q2 is 0, 1, 2, 3, or 4.

In certain embodiments, q2 is 0. In certain embodiments, q2 is 1. Incertain embodiments, q1 is 2. In certain embodiments, q2 is 3. Incertain embodiments, q2 is 4.

In certain embodiments, R^(q2) is hydrogen. In certain embodiments,R^(q2) is optionally substituted aliphatic. In certain embodiments,R^(q2) is optionally substituted C₁₋₆ alkyl.

In certain embodiments, R^(t) is optionally substituted aliphatic andR^(q2) is hydrogen.

In certain embodiments, -L²-F¹⁸ is of the formula:

In certain embodiments, wherein -L²-F¹⁸ is of Formula (iii-b4):

wherein

L³ is a bond, optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted arylene, optionally substitutedheteroarylene, or optionally substituted heterocyclylene;

R^(N1) is independently hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl, oroptionally substituted heteroaryl, or a nitrogen protecting group; and

R^(Z) is independently hydrogen, optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substituted aryl, oroptionally substituted heteroaryl, wherein RZ comprise ¹⁸F.

In certain embodiments, wherein -L²-F¹⁸ is of Formula (iii-b4-a):

In certain embodiments, R^(Z) is an optionally substituted thiol.

In certain embodiments, -L²-F¹⁸ is of Formula (iii-c):

wherein

Y is a ligand capable of chelating to a pharmaceutically acceptablemetal complex comprising F¹⁸; and

L⁴ is a bond, optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted arylene, optionally substitutedcycloalkylene, optionally substituted heteroarylene, or optionallysubstituted heterocyclylene.

As generally defined herein, Y is a ligand capable of chelating to apharmaceutically acceptable metal complex comprising F¹⁸. As usedherein, a ligand refers to an ion or molecule (functional group) thatbinds to a central metal atom to form a coordination complex. Thebonding between metal and ligand generally involves formal donation ofone or more of the ligand's electron pairs. The nature of metal-ligandbonding can range from covalent to ionic. Exemplary monodentate ligandsinclude, but are not limited to, CO, organonitriles (e.g., CH₃CN,CH₃CH₂CN), monosubstituted amines, disubstituted amines, trisubstitutedamines, heterocyclyls (e.g., pyridine, piperidine), dialkylcyanamides,triphenylphosphine oxide, THF, DMF, or NMF. Exemplary bidentate ligandsinclude, but are not limited to, 1,5-cyclooctadiene, norbornadiene,1,2-ethylenediamine, tetramethylethylenediamine, 1,2-dimethoxyethane,diglyme, or 2,5-dithiahexane. Exemplary tridentate ligands include, butare not limited to, conjugated cyclic triene (e.g., cycloheptatriene),conjugated acyclic triene, arenes (e.g., benzene, toluene, xylene,mesitylene, naphthalene), tetraazamacrocyles (e.g.,tetraazacyclododecane), polyamines (e.g., diethylenetriamine), andtrithiocylononane. In certain embodiments, the ligand is a polydentateligand. In certain embodiments, the ligand comprises1,4,7-triazacyclononane-triacetic acid (NOTA),1,4,7,10-tetraazacyclododecane-tetraacetic acid (DOTA), ortriazacyclononane-phosphinate (TRAP).

The phrase “pharmaceutically acceptable” means that the metal complex issuitable for administration to a subject. In certain embodiments, themetal complex is a halide metal complex. In certain embodiments, themetal is a pharmaceutically acceptable metal. In certain embodiments,the metal is IIA or IIIA group metal. In certain embodiments, the metalis an early transition metal. In certain embodiments, the metal is A1.

In certain embodiments, L⁴ is a bond. In certain embodiments, L⁴ is anoptionally substituted aliphatic. In certain embodiments, L⁴ is anoptionally substituted heteroaliphatic.

In certain embodiments, -L²-F¹⁸ is of Formula (iii-c1):

wherein

M is a pharmaceutically acceptable metal;

each of L^(a), L^(b), and L^(c) is independently optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted arylene, optionally substituted cycloalkylene, optionallysubstituted heteroarylene, or optionally substituted heterocyclylene;and

“---” indicates a coordination bond or absend, as valency permits.

In certain embodiments, L^(a) is optionally substituted aliphatic. Incertain embodiments, L^(a) is optionally substituted heteroaliphatic. Incertain embodiments, L^(a) is optionally substituted heteroalkylene. Incertain embodiments, L^(a) is —(CH₂)₁₋₃—C(═O)O—, wherein the chelationpoint to M is O. In certain embodiments, L^(a) is —CH₂—C(═O)O—. Incertain embodiments, L^(a) is —(CH₂)₁₋₃—C(═O)OH, wherein the chelationpoint to M is O. In certain embodiments, L^(a) is —CH₂—C(═O)OH.

In certain embodiments, L^(b) is optionally substituted aliphatic. Incertain embodiments, L^(b) is optionally substituted heteroaliphatic. Incertain embodiments, L^(b) is optionally substituted heteroalkylene. Incertain embodiments, L^(b) is —(CH₂)₁₋₃—C(═O)O— wherein the chelationpoint to M is O. In certain embodiments, L^(b) is —CH₂—C(═O)O—. Incertain embodiments, L^(b) is —(CH₂)₁₋₃—C(═O)OH, wherein the chelationpoint to M is O. In certain embodiments, L^(b) is —CH₂—C(═O)OH.

In certain embodiments, L^(c) is optionally substituted aliphatic. Incertain embodiments, L^(c) is optionally substituted heteroaliphatic. Incertain embodiments, L^(c) is optionally substituted heteroalkylene. Incertain embodiments, L^(c) is —(CH₂)₁₋₃—C(═O)O— wherein the point ofattachment to M is O. In certain embodiments, L^(c) is —CH₂—C(═O)O—. Incertain embodiments, L^(c) is —(CH₂)₁₋₃—C(═O)OH, wherein chelation pointto M is O. In certain embodiments, L^(c) is —CH₂—C(═O)OH.

As generally defined herein, “---” indicates the chelation formedbetween M and the ligand, as valency permits. In certain embodiments, Mforms one chelating bond with the ligand. In certain embodiments, Mforms two chelating bonds with the ligand. In certain embodiments, Mforms three chelating bonds with the ligand.

In certain embodiments, -L²-F¹⁸ is of Formula (iii-c2):

In certain embodiments, the radioactive protein is one of the followingformulae:

In certain embodiments, the linker L² is of one of the followingformulae:

wherein R^(s), R^(t), R^(c1), R^(c2), and m are as defined herein.

In certain embodiments, provided herein is a radioactive protein ofFormula (III)

wherein

L¹ is as defined herein; and

R³ comprises a ligand capable of chelating to a pharmaceuticallyacceptable radioactive metal complex.

In certain embodiments, L¹-R³ is of the following formula:

wherein R^(Z2) is optionally substituted aliphatic, optionallysubstituted heteroaliphatic, optionally substituted aryl, or optionallysubstituted heteroaryl, or a nitrogen protecting group; wherein R^(z2)comprises a ligand capable of chelating to a pharmaceutically acceptableradioactive metal complex.

In certain embodiments, R³ comprises mono-dentecate ligand. In certainembodiments, R³ comprises a polydentecate ligand. In certainembodiments, R³ comprises 1,4,7-triazacyclononane-triacetic acid (NOTA),1,4,7,10-tetraazacyclododecane-tetraacetic acid (DOTA), ortriazacyclononane-phosphinate (TRAP).

In certain embodiments, the metal is ⁶⁴Cu²⁺. In certain embodiments, themetal is ⁶⁸Ga³⁺.

In certain embodiments, the radioactive protein is one of the followingformulae:

In certain embodiments, provided herein is a radioactive protein ofFormula (IV)

wherein

L¹ is a linker comprising at least four amino acids formed by enzymaticconjugation between two enzyme recognition sequences;

R⁴ comprises a radioactive optionally substituted carbohydrate; and

R⁴ is linked to the C-terminus of the adjacent amino acid in L¹.

As generally defined herein, R⁴ comprises a radioactive optionallysubstituted carbohydrate. In certain embodiments, R⁴ comprises aradioactive optionally substituted glucose. In certain embodiments, R⁴comprises a radioactive glucose comprising ¹⁸F. In certain embodiments,R⁴ comprises an optionally substituted glucose comprising ¹⁸F. Incertain embodiments, R⁴ is linked to the C-terminus of the adjacentamino acid in L¹. In certain embodiments, R⁴ is linked to the side chainof the adjacent amino acid in L¹.

In certain embodiments, R⁴ is of Formula (iv):

wherein

v is 1, 2, 3, 4, or 5; and

each of R^(s5), R^(s6), R^(s7), and R^(s8) is independently hydrogen,optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, or optionally substitutedheteroaryl, or an oxygen protecting group.

In certain embodiments, R^(s5) is independently hydrogen. In certainembodiments, R^(s5) is optionally substituted aliphatic. In certainembodiments, R^(s5) is optionally substituted alkyl. In certainembodiments, R^(s5) is an oxygen protectin group. In certainembodiments, R^(s5) is acyl (e.g. acetyl).

In certain embodiments, R^(s6) is independently hydrogen. In certainembodiments, R^(s6) is optionally substituted aliphatic. In certainembodiments, R^(s6) is optionally substituted alkyl. In certainembodiments, R^(s6) is an oxygen protectin group. In certainembodiments, R^(s6) is acyl (e.g. acetyl).

In certain embodiments, R^(s7) is independently hydrogen. In certainembodiments, R^(s7) is optionally substituted aliphatic. In certainembodiments, R^(s7) is optionally substituted alkyl. In certainembodiments, R^(s7) is an oxygen protectin group. In certainembodiments, R^(s7) is acyl (e.g. acetyl).

In certain embodiments, R^(s8) is independently hydrogen. In certainembodiments, R^(s8) is optionally substituted aliphatic. In certainembodiments, R^(s8) is optionally substituted alkyl. In certainembodiments, R^(s8) is an oxygen protectin group. In certainembodiments, R^(s8) is acyl (e.g. acetyl).

In certain embodiments, R⁴ is of the formula:

In certain embodiments, the radioactive protein is of the followingformula:

wherein R^(s5), R^(s6), R^(s7), and R^(s8) are as defined herein; andL^(G) is optionally substituted aliphatic or optionally substitutedheteroaliphatic.

In certain embodiments, L^(G) is optionally substituted aliphatic. Incertain embodiments, L^(G) is optionally substituted C₁₋₁₀ alkyl. Incertain embodiments, L^(G) is optionally substituted heteroaliphatic. Incertain embodiments, L^(G) is of the formula:

wherein e indicates the point of attachment to oxygen and f indicatesthe point of attachment to the alpha carbon of the amino acid.

In certain embodiments, the radioactive protein is of the followingformula:

In certain embodiments,

is an antibody, a nuclear factor, a neuropeptide, a receptor protein, anenzyme, a structural protein, or a fragment thereof. In certainembodiments,

is an antibody or a fragment thereof. In certain embodiments,

is VHH or a fragment thereof.

Synthesis of Intermediates and Radiolabeled Proteins

The oxime compounds of Formula (b-2-a2)

can be prepared from optionally substituted tetrazine-aminooxy and aradiolabeled optionally substituted aldehyde or optionally substitutedketone of the formula R^(as)—CO—R^(bs), wherein R^(G1) is as definedherein; each of R^(as) and R^(bs) is independently hydrogen, optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted aryl, optionally substituted heteroaryl, oroptionally substituted heterocyclyl; provided R^(as) and R^(bs) are notboth hydrogen (Scheme S1).

In certain embodiments, R^(as) is an optionally substituted carbohydrategroup or a fragment thereof. In certain embodiments, R^(as) is anoptionally substituted glucose or a fragment thereof. In certainembodiments, the reaction is carried out in the presence of a catalyst.In certain embodiments, the catalyst is m-phenylenediamine,p-phenylenediamine, or p-anisidine. In certain embodiments, the catalystis m-phenylenediamine. In certain embodiments, the molar ratio of theoptionally substituted tetrazine-aminooxy to the catalyst is from about10:1 to 1:10. In certain embodiments, the molar ratio of the optionallysubstituted tetrazine-aminooxy to the catalyst is from about 1:1 to 1:8.In certain embodiments, the molar ratio of the optionally substitutedtetrazine-aminooxy to the catalyst is from about 1:1 to 1:6. In certainembodiments, the molar ratio of the optionally substitutedtetrazine-aminooxy to the catalyst is from about 1:2 to 1:4. In certainembodiments, the molar ratio of the optionally substitutedtetrazine-aminooxy to the catalyst is about 1:4.

In certain embodiments, R^(as) is an optionally substituted carbohydrategroup or a fragment thereof, provided R^(as) comprises ¹⁸F. In certainembodiments, R^(as) is an optionally substituted glucose or a fragmentthereof. In certain embodiments, R^(as) is ¹⁸F-FDG of a fragmentthereof.

As provided in Scheme S1, the resulting oxime product can be easilypurified from the reaction mixture to the change in hydrophilicity.

In certain embodiments of Scheme S1, the excess of tetrazine-aminooxycan be captured by reacting with another water soluble carbohydrate. Incertain embodiments, the water soluble carbohydrate is glucosamine6-sulfate.

The compound of Formula (b-2-b)

can be prepared from reacting an optionally substituted tetrazinecomprising a nucleophic group with an electrophile comprising ¹⁸F suchas ¹⁸F-SFB. Exemplary synthesis of Formula (b-2-b) is provided in SchemeS2.

In certain embodiments, the Nu is an amino group. In certainembodiments, the electrophile is an optionally substitutedN-succinimidyl comprising ¹⁸F. In certain embodiments, the optionallysubstituted N-succinimidyl is ¹⁸F-SFB of the formula

Exemplary synthesis of ¹⁸F-SFB can be found in FIG. 11.

The radioactive protein of Formula (II) can be prepared from a modifiedprotein of Formula (I) with a compound of Formula (b): ¹⁸F—R² (b),wherein R² is a reactive group capable of undergoing the click chemistryreaction (Scheme S3):

In certain embodiments, R¹ is the first click chemistry handle and R² isthe second click chemistry handle. In certain embodiments, R² is thefirst click chemistry handle and R¹ is the second click chemistryhandle.

Click chemistry should be modular, wide in scope, give high chemicalyields, generate inoffensive byproducts, be stereospecific, bephysiologically stable, exhibit a large thermodynamic driving force(e.g., >84 kJ/mol to favor a reaction with a single reaction product),and/or have high atom economy. Several reactions have been identifiedwhich fit this concept:

(1) The Huisgen 1,3-dipolar cycloaddition (e.g., the Cu(I)-catalyzedstepwise variant, often referred to simply as the “click reaction”; see,e.g., Tornoe et al., Journal of Organic Chemistry (2002) 67: 3057-3064).Copper and ruthenium are the commonly used catalysts in the reaction.The use of copper as a catalyst results in the formation of1,4-regioisomer whereas ruthenium results in formation of the1,5-regioisomer;

(2) Other cycloaddition reactions, such as the Diels-Aldercycloaddition;

(3) Nucleophilic addition to small strained rings like epoxides andaziridines;

(4) Nucleophilic addition to activated carbonyl groups; and

(4) Addition reactions to carbon-carbon double or triple bonds.

In certain embodiments, the click chemistry is a Diels-Aldercycloaddition. Exemplary Diels-Alder cycloadditions can be found in U.S.Patent Publication No. 20130266512, which is incorporated by referenceherein;

The radioactive protein of Formula (III) can be prepared from a compoundcomprising an aminooxy moiety with an optionally substituted aldehyde(Scheme S4):

The radioactive protein of Formula (IV) can be prepared from a compoundcomprising an aminooxy moiety with an optionally substituted aldehyde oran optionally substituted ketone (Scheme S5), wherein L^(G) is asdefined herein.

In certain embodiments, the aldehyde is an optionally substitutedcarbohydrate comprising an aldehyde group or is capable of forming onethrough isomerism. In certain embodiments, the optionally substitutedaldehyde is an optionally substituted monosaccharide. In certainembodiments, the optionally substituted aldehyde is optionallysubstituted glucose, optionally substituted glyceraldehyde, oroptionally substituted galactose. In certain embodiments, the optionallysubstituted aldehyde is optionally substituted glucose.

In certain embodiments, the catalyst is m-phenylenediamine (mPDA),o-phenylenediamine, p-phenylenediamine, o-aminophenol, m-aminophenol,p-aminophenol, o-aminobenzoic acid, 5-methoxyanthranilic acid,3,5-diaminobenzoic acid or aniline. In certain embodiments, the catalystis m-phenylenediamine (mPDA).

In certain embodiments, the radioactive optionally substitutedcyclooctene is synthesized by a nucleophilic reaction with an ¹⁸F anionwith a substituted cyclooctene comprising a leaving group LG (SchemeS6), wherein LG is as defined herein and L⁶¹ is optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted arylene, optionally substituted heteroarylene, or optionallysubstituted heterocyclylene.

In certain embodiments, L⁶¹ is optionally substituted hereoaliphatic. Incertain embodiments, L⁶¹ is straight chain heteroaliphatic. In certainembodiments, L⁶¹ is —O—C₁₋₈alkylene. In certain embodiments, L⁶¹ is—O—(CH₂)₁₋₈.

In certain embodiments, the radioactive optionally substitutedcyclooctene is synthesized as shown in Scheme S6-a, wherein L⁶¹ is asdefined herein.

In certain embodiments, the ¹⁸F⁻ anion is from an inorganic saltcomprising ¹⁸F⁻ anion. In certain embodiments, the ¹⁸F⁻ anion is from ametal salt comprising ¹⁸F⁻ anion. In certain embodiments, the ¹⁸F⁻ anionis from IA, IIA, or IIIA metal fluoride. In certain embodiments, the¹⁸F⁻ anion is from transition metal complex comprising ¹⁸F⁻.

In certain embodiments, the enzymatic conjugation is a modificationusing a formylglycine generating enzyme (FGE). In certain embodiments,the protein is an antibody. In certain embodiments, the enzyme is FGE.In certain embodiments, the FGE recognition sequence is CXPXR. Incertain embodiments, the FGE recognition sequence is LCTPSRGSLFTGR. Incertain embodiments, the radioactive protein is prepared according toScheme E1.

It is to be understood that the —CHO group generated from the FGEmodification can undergo any suitable reaction to incorporate aradioactive label, for example, a click chemistry handle, a radioactivecarbohydrate, or a ligand capable of chelating to a pharmaceuticallyacceptable radioactive metal complex. Exemplary transformations such asreacting with hydrazine or hydroxylamine are shown in the Scheme E1.

In certain embodiments, the enzymatic conjugation is a modificationusing sialyltransferases. In certain embodiments, the protein is a cellsurface polypeptide. In certain embodiments, the protein is a glycan. Anexemplary sialylation is shown in Scheme E2, wherein R⁴ is as definedherein.

In certain embodiments, R⁴ comprises radioactive optionally substitutedglucose. In certain embodiments, R⁴ comprises ¹⁸F-FDG. In certainembodiments, R⁴ comprises radioactive optionally substituted aldolase.In certain embodiments, R⁴ comprises radioactive optionally substitutedmannose.

In certain embodiments, the enzymatic conjugation is a modificationusing phosphopantetheinyltransferases (PPTases). In certain embodiments,the protein is peptide carrier protein (PCP). In certain embodiments,the protein is acyl carrier protein (ACP). In certain embodiments, thePPT recognition sequence comprises a serine residue. In certainembodiments, the PPTase recognition sequence is DSLEFIASKLA,VLDSLEFIASKLA, or GSQDVLDSLEFIASKLA. In certain embodiments, thephosphopantetheinyltransferase is Sfp. An exemplary modification usingPPTase is shown in Scheme E3, wherein R^(F) is as defined herein.

In certain embodiments, the enzymatic conjugation is a modificationusing polypeptidyltransferases (OGTases). In certain embodiments, theprotein is nuclear pore protein. In certain embodiments, the OGTase isUDP-Glc-NAc. In certain embodiments, the OGTase recognition sequencecomprises a serine residue or threonine residue. Exemplary modificationsusing polypeptidyltransferases are shown in Scheme E4, wherein R^(F) isas defined herein.

In certain embodiments, the enzymatic conjugation is a modificationusing transglutaminase (TGases). In certain embodiments, the protein isan antibody. In certain embodiments, the TGase recognition sequencecomprises a glutamine (Q) residue. In certain embodiments, the TGaserecognition sequence comprises XXQXX. In certain embodiments, theprotein recognition sequence is GGGSLLQG, PNPQLPF, PKPQQFM, or GQQQLG.In certain embodiments, the protein recognition sequence comprises alysine (K) residue. In certain embodiments, the protein recognitionsequence is MRHKGS. An exemplary modification using TGases is shown inScheme E5, wherein R^(F) is as defined herein.

In certain embodiments, the enzymatic conjugation is a modificationusing protein farnesyltransferase (PFTase). In certain embodiments, theprotein is an antibody. In certain embodiments, the PFTase recognitionsequence comprises CaaX, wherein each instance of a is independently analiphatic amino acid and X is as defined herein. Exemplary modificationsusing PFTases are shown in Scheme E6, wherein R^(F) is as definedherein.

In certain embodiments, the enzymatic conjugation is a modificationusing biotin ligases. In certain embodiments, the protein is anantibody. In certain embodiments, the biotin ligase recognition sequencecomprises lysine (K). In certain embodiments, the biotin ligaserecognition sequence comprises GLNDIFEAQKIEWHE. In certain embodiments,the enzyme is E. coli biotin ligase, BirA. An exemplary modificationusing biotin ligases is shown in Scheme E7, wherein R^(F) is as definedherein.

In certain embodiments, the enzymatic conjugation is a modificationusing lipoic acid ligases (Lp1As). In certain embodiments, the proteinis an antibody. In certain embodiments, the protein is a growth factorreceptor. In certain embodiments, the Lp1A recognition sequencecomprises GFEIDKVWYDLDA. In certain embodiments, the enzyme is E. coliLp1. An exemplary modification using Lp1As is shown in Scheme E8,wherein R^(F) is as defined herein.

In certain embodiments, the enzymatic conjugation is a modificationusing N-myristoyltransferase (NMT). In certain embodiments, the proteinis an antibody. In certain embodiments, the protein is a tyrosinekinase. In certain embodiments, the protein is a HIV-1 matrix protein.In certain embodiments, the protein is a HIV Gag. In certainembodiments, the protein is an ADP-ribosylating factor. In certainembodiments, the NMT recognition sequence comprises GXXXS/T, wherein Xis any amino acid. An exemplary modification using NMT is shown inScheme E9, wherein R^(F) is as defined herein.

In certain embodiments, R^(F) is a reactive group capable of undergoinga click chemistry reaction. In certain embodiments, R^(F) is R¹ asdefined herein. In certain embodiments, R^(F) is optionally substitutedtetrazine. In certain embodiments, R^(F) is optionally substitutedtetrazine comprising ¹⁸F. In certain embodiments, R^(F) is optionallysubstituted tetrazine comprising ¹⁸F-FDG or a fragment thereof. Incertain embodiments, R^(F) is optionally substituted tetrazinecomprising ¹⁸F-SFB or a fragment thereof. In certain embodiments, R^(F)is optionally substituted cyclooctene. In certain embodiments, R^(F) isoptionally substituted trans-cyclooctene. In certain embodiments, R^(F)is optionally substituted trans-cyclooctene comprising ¹⁸F. In certainembodiments, R^(F) is comprises a ligand capable of chelating to apharmaceutically acceptable radioactive metal complex. In certainembodiments, R^(F) is R³ as defined herein. In certain embodiments,R^(F) comprises a ligand capable of chelating to a pharmaceuticallyacceptable metal complex comprising F¹⁸. In certain embodiments, R^(F)is Y as defined herein. In certain embodiments, R^(F) comprises aradioactive optionally substituted carbohydrate. In certain embodiments,R^(F) is R⁴ as defined herein. In certain embodiments, R^(F) comprises¹⁸F-FDG or a fragment thereof.

Kits

Some aspects of this invention provide kits useful for labeling proteinswith radioactive agents and/or hydrophilic polymers, and/or forgenerating radiolabeled sortase substrate peptides.

In some embodiments, the kit comprises a radiolabeled protein generatedaccording to an inventive method provided herein. In some embodiments,the kit comprises (i) a single domain antibody comprising a sortaserecognition sequence, (ii) a sortase substrate comprising a first clickchemistry handle, and (iii) a hydrophilic polymer conjugated to a secondclick chemistry handle. In some embodiments, the kit comprises (i) afirst single domain antibody comprising a first click chemistry handle,and (ii) a second single domain antibody comprising a second clickchemistry handle. In some embodiments, the kit comprises (i) a firstsingle domain antibody comprising a sortase recognition sequence, (ii) afirst sortase substrate, wherein the first sortase substrate comprises afirst click chemistry handle, (iii) a second single domain antibodycomprising a sortase recognition sequence, and (iv) a second sortasesubstrate, wherein the second sortase substrate comprises a second clickchemistry handle. In some embodiments, the kit further comprises aradionuclide. In some embodiments, the radionuclide is carbon-11,carbon-14, nitrogen-13, oxygen-15, fluorine-18, rubidium-82, copper-61,copper-62, copper-64, yttrium-86, gallium-68, zirconium-89, oriodine-124. In some embodiments, the kit further comprises a sortase Afrom Staphylococcus aureus (SrtAaureus), sortase A from Streptococcuspyogenes (SrtApyogenes), sortase B from S. aureus (SrtBaureus), sortaseB from Bacillus anthracis (SrtBanthracis), or sortase B from Listeriamonocytogenes (SrtBmonocytogenes).

In some embodiments, the kit further comprises a buffer or reagentuseful for carrying out a sortase-mediated transpeptidation reaction.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. The examples describedin this application are offered to illustrate the methods, compositions,and systems provided herein and are not to be construed in any way aslimiting their scope.

Example 1. Enzyme-Mediated Dual-Labeling of Antibodies for MultimodalImaging and to Manipulate their Characteristics

Structures of VHHs show that their C-terminus is positioned away fromthe antigen binding site^([13]). Therefore a chemical approach waschosen to link two fully functional VHHs via their C-termini to ensurethat their antigen binding capacity would not be compromised bymodification of one of the N-termini in the resulting fusion, and thatthe two binding sites thus created would be equivalent, which may bemore difficult to ascertain for genetic fusions.

Four sortase substrates were designed and synthesized for production ofdimers or PEGylated VHHs (FIG. 1). The substrates either contain twobioorthogonal handles or a handle and a flourophore. TheAlexa647-labeled substrate was designed such that the reaction productscould be used in FACS experiments to estimate relative in vitro bindingaffinities. The Texas Red-modified substrate was designed to enabletwo-photon microscopy and to estimate relative in vivo bindingaffinities; the TCO-modified substrate was produced to allow rapidinstallation of a tetrazine-functionalized radioactive tag for PET, inthis case an isotopically labelled F-labeled-tetrazine radioactive tagfor PET (t1/2=110 min. 18F t1/2<2 h). The dimers were produced asexemplified in FIG. 2. For DC8 (anti Class II), splenocytes were stainedwith different concentrations of monomers and dimers and analyzed byFACS. For DC13 (anti CD11b), the CD11b⁺ mutuDC dendritic cell line wasused. For Class II MHC and CD11b, VHH dimers bind approximately 3.3 and2.3-fold better than their corresponding monomers respectively (FIG. 3).The in vivo binding characteristics of the VHH dimers versus monomerswere evaluated. Due to the more abundant expression of Class II MHCmolecules on splenocytes compared to that of CD11b, the in vivo bindingaffinity for anti-Class II dimers was explored. Mice were injected i.v.with equal amounts of monomers and dimers (0.25 nmol) of DC8. 2 h p.i.,mice were euthanized and lymphoid organs were excised for examination.Monomers and dimers yielded different staining patterns (FIG. 3B), withthe dimer showing interspersed yet more pronounced staining than themonomer, corroborating the FACS results. To render monomers and dimerssuitable for immuno-PET, dimer VHHs were produced using substrate 4. Toproduce the ¹⁸F-tetrazine, a method was applied that was automated inits key steps to minimize operator radiation exposure in the course ofpreparation (FIG. 4A). The ¹⁸F-labeled anti Class II and anti CD11bdimers were produced and applied them to PET imaging (FIG. 4). PETshowed that both the dimers stained lymphoid organs (FIG. 4). The antiClass II MHC dimer showed stronger staining of lymphoid organs,particularly the spleen, compared to the anti CD11b dimer (FIGS. 4D and4F). This may be attributed to the fact that in spleen there are fewerCD11b⁺ cells compared to Class II MHC⁺ cells and less CD11b expressedper cell. Specificity was established by blocking the targets of theseVHHs by introduction of unlabeled VHHs as competitors prior to imaging.PET imaging conducted 2 h p.i. of ¹⁸F-labeled VHHs showed effectiveblocking of signal in the lymphoid organs, further underlining thespecificity of the signals (FIG. 5C). To further confirm the specificityof the dimers in the absence of competing VHHs, MHC II^(−/−) andCD11b^(−/−) mice were imaged. No PET signals were detected in lymphoidorgans, further confirming the specificity of both dimers (FIGS. 4E-4G).Monomers and dimers do not differ in uptake in kidneys and intestine,organs commonly targeted non-specifically by VHHs in the course of theirclearance^([14]). These experiments helped set the stage to exploreability of VHH dimers to image tumors. Two types of tumors were imagedwith the developed bivalent VHHs by engraftment of mice with the B16melanoma or the pancreatic tumor cell line panc02. Both the dimersreadily detected the lymphoid organs as well as the B16 melanoma tumoror the panc02 graft. Upon excision, the panc-tumors were no more than˜1-2 mm in diameter, showing the method to be sensitive. Postmortembiodistribution analysis correlated well with the FACS and two-photonresults as well (FIGS. 5A-5B). It was examined whether circulatoryhalf-life could be manipulated to further improve the signal to noiseratio. PEGylation of VHHs results in increased circulatory half-life,where the increase correlates with the size of the attached PEGgroup^([15]). VHHs sortagged with substrate 2 or 3 were reacted withDBCO-functionalized PEG (5 kDa, 10 kDa or 20 kDa) to yield the finalPEGylated VHHs (FIGS. 2E-2F). B6 mice received 5 μg of PEGylated VHH andwere euthanized 3 h later, followed by dissection of lymph nodes andspleen for FACS and two-photon microscopy. FACS showed a significantincrease in staining of the PEGylated DC8-Alexa647 versus thenon-PEGylated DC8. An approximate 30%, 100%, and 220% increase instaining was observed for 5 kDa, 10 kDa, or 20 kDa PEG-conjugated DC8,respectively, establishing a correlation between the size of theattached PEG moiety and their staining efficiency (FIG. 3D). WhenDC8-Alexa647-PEG-20 kDa were injected, which showed the strongestbinding in vivo, into a MHC II^(−/−) mouse, FACS analysis showed nostaining in the spleen (FIG. 3D). This confirmed that PEGylation doesnot affect the specificity of the VHHs.

The PEGylated VHHs were explored for their suitability in PET imaging.VHH DC8 was PEGylated and modified with an ¹⁸F radionuclide. PEGylatedVHHs showed increased staining of the lymphoid organs, confirming FACSand two-photon microscopy (FIG. 5D). The longer circulatory half-life ofPEGylated VHHs may increase the likelihood of a VHH binding to itstarget, as long as target accessibility is not compromised byPEGylation. The ¹⁸F-labeled-DC8-PEG-20 kDa had the highest PET signal inblood at the time of imaging (3 h p.i) (FIG. 5D). While the larger 20kDa PEG can significantly increase the staining efficiency in lymphoidorgans, it can delay circulatory clearance, a factor to consider whenisotopes with short half-lives, such as 18F or 11C, are being used.However, a 10 kDa or a 5 kDa PEG-modified VHH can still significantlyincrease staining efficiency and yet be rapidly cleared. For labeling ofVHHs, the use of isotopes with longer half-lives, such as ⁸⁹Zr or ⁶⁴Cu,the use of a 20 kDa PEG moiety may improve staining.

In summary, the methods provided herein may produce site-specificallyPEGylated or bivalent single domain antibodies equipped with afluorophore and/or radionuclide (¹⁸F) for the different imagingmodalities. The reaction conditions are compatible with full retentionof biological activity of the fusion partners. By cytofluorimetry,dimers bind approximately 3 times more avidly to their targets.Two-photon microscopy demonstrated that Texas Red-labeled bivalentsingle domain derivatives bind more efficiently to their targets.PEGylated fluorescent VHHs showed improved staining in vivo, with largerPEG substituents giving stronger signals in FACS. In immuno-PETexperiments, bivalent ¹⁸F-labeled VHHs better stained lymphoid organs invivo than monomers. ¹⁸F-labeled PEGylated VHHs showed improved staining,with higher MW PEG substituents giving stronger signals. Finally,immuno-PET of two different models of tumor-bearing mice, showed thatthe DC8-dimer and DC13-dimer detected not only lymphoid organs, but alsoshowed the location of ectopic melanoma and pancreatic tumor grafts assmall as about 1 mm in size. Overall, these data provide support thatderivatives of single domain antibodies are a valuable addition to thecurrent imaging and radiodiagnostic approaches.

REFERENCES

-   [1] T. Olafsen, S. J. Sirk, S. Olma, C. K.-F. Shen, A. M. Wu, Tumor    Biol. 2012, 33, 669-677.-   [2] E. J. Lipson, W. H. Sharfman, C. G. Drake, I. Wollner, J. M.    Taube, R. A. Anders, H. Xu, S. Yao, A. Pons, L. Chen, et al., Clin.    Cancer Res. Off J. Am. Assoc. Cancer Res. 2013, 19, 462-468.-   [3] T. R. Simpson, F. Li, W. Montalvo-Ortiz, M. A. Sepulveda, K.    Bergerhoff, F. Arce, C. Roddie, J. Y. Henry, H. Yagita, J. D.    Wolchok, et al., J. Exp. Med. 2013, 210, 1695-1710.-   [4]R. Boellaard, M. J. O'Doherty, W. A. Weber, F. M. Mottaghy, M. N.    Lonsdale, S. G. Stroobants, W. J. G. Oyen, J. Kotzerke, O. S.    Hoekstra, J. Pruim, et al., Eur. J. Nucl. Med. Mol. Imaging 2010,    37, 181-200.-   [5] M. Rashidian, E. J. Keliher, A. M. Bilate, J. N. Duarte, G. R.    Wojtkiewicz, J. T. Jacobsen, J. Cragnolini, L. K. Swee, G. D.    Victora, R. Weissleder, et al., Proc. Natl. Acad. Sci. U.S.A. 2015,    DOI 10.1073/pnas.1502609112.-   [6] R. Tavare, M. N. McCracken, K. A. Zettlitz, S. M. Knowles, F. B.    Salazar, T. Olafsen, O. N. Witte, A. M. Wu, Proc. Natl. Acad. Sci.    2014, 111, 1108-1113.-   [7] M. Rashidian, E. J. Keliher, M. Dougan, P. K. Juras, M.    Cavallari, G. R. Wojtkiewicz, J. T. Jacobsen, J. G. Edens, J. M. J.    Tas, G. Victora, et al., ACS Cent. Sci. 2015, 150603073029009.-   [8] E. C. Dijkers, T. H. Oude Munnink, J. G. Kosterink, A. H.    Brouwers, P. L. Jager, J. R. de Jong, G. A. van Dongen, C. P.    Schrider, M. N. Lub-de Hooge, E. G. de Vries, Clin. Pharmacol. Ther.    2010, 87, 586-592.-   [9] M. Rashidian, E. J. Keliher, A. M. Bilate, J. Doarte, G.    Wojtkiewicz, J. Jacobsen, G. Victora, R. Weissleder, H. L. Ploegh,    Proc. Natl. Acad. Sci. U.S.A. 2015.-   [10] T. De Meyer, S. Muyldermans, A. Depicker, Trends Biotechnol.    2014, 32, 263-270.-   [11] P. Holliger, T. Prospero, G. Winter, Proc. Natl. Acad. Sci.    U.S.A. 1993, 90, 6444-6448.-   [12] H. Schellekens, W. E. Hennink, V. Brinks, Pharm. Res. 2013,    30,1729-1734.-   [13] E. De Genst, K. Silence, K. Decanniere, K. Conrath, R.    Loris, J. Kinne, S. Muyldermans, L. Wyns, Proc. Natl. Acad. Sci.    U.S.A. 2006, 103, 4586-4591.-   [14] V. Cortez-Retamozo, M. Lauwereys, G. Hassanzadeh Gh, M.    Gobert, K. Conrath, S. Muyldermans, P. De Baetselier, H. Revets,    Int. J. Cancer J. Int. Cancer 2002, 98, 456-462.-   [15] Y. Vugmeyster, C. A. Entrican, A. P. Joyce, R. F.    Lawrence-Henderson, B. A. Leary, C. S. Mahoney, H. K. Patel, S. W.    Raso, S. H. Olland, M. Hegen, et al., Bioconjug. Chem. 2012, 23,    1452-1462.

Example 2. Materials and Methods Synthesis of(Gly)₃-PEG₁₂-Cys(TCO)-PEG₅-Lys(azide)

The peptide (Gly)₃-PEG₁₂-Cys-PEG₅-Lys(azide) was synthesized by standardsolid phase peptide synthesis. Maleimide-TCO (from Conju-bio) wasdissolved in 0.05 M NaHCO₃ buffer pH 8.3. The peptide was added and leftto stir at room temperature for 1 h until LC-MS indicated near-completeconversion to the product. The solution was filtered and purified byreverse phase-HPLC with a semi-preparative column (Phenomenex, C₁₈column, Gemini, 5 μm, 10×250 mm) at a flow rate of 5.0 mL/min.; solventA: 0.1% formic acid in H₂O, solvent B: 0.1% formic acid in CH₃CN.Product eluted at 35-40% solvent B. Fractions containing pure productwere collected and lyophilized. LC-MS calculated for C₈₃H₁₅₁N₁₄O₃₄S[M+H]⁺ 1920.0, found 1919.0.

Synthesis of (Gly)₃-PEG₁₂-Cys(Texas Red)-PEG₅-Lys(azide)

The peptide (Gly)₃-PEG₁₂-Cys-PEG₅-Lys(azide) was synthesized by standardsolid phase peptide synthesis and was dissolved in 0.05 M NaHCO₃ bufferpH 8.3. Maleimide-Texas Red (from Vector Labs) was dissolved in DMSO andthen was added to the solution and left to stir at room temperature for1 h until LC-MS indicated near-complete conversion to the product. Thesolution was filtered and purified by reverse phase-HPLC with asemi-preparative column (Phenomenex, C₁₈ column, Gemini, 5 am, 10×250mm) at a flow rate of 5.0 mL/min.; solvent A: 0.1% TFA in H₂O, solventB: 0.1% TFA in CH₃CN. Product eluted at 40-45% solvent B. Fractionscontaining pure product were collected and lyophilized. LC-MS calculatedfor C₉₂H₁₄₂N₁₅O₃₂S₃[M+H]⁺ 2064.9, found 2063.9.

Synthesis of (Gly)₃-PEG₁₂-Cys(Alexa647)-PEG₅-Lys(azide)

The peptide (Gly)₃-PEG₁₂-Cys-PEG₅-Lys(azide) was synthesized by standardsolid phase peptide synthesis. Maleimide-Alexa647 (from Life Technology)was dissolved in 0.05 M NaHCO₃ buffer pH 8.3. The peptide was added andleft to stir at room temperature for 1 h until LC-MS indicatednear-complete conversion to the product. The solution was filtered andpurified by reverse phase-HPLC with a semi-preparative column(Phenomenex, C₁₈ column, Gemini, 5 μm, 10×250 mm) at a flow rate of 5.0mL/min.; solvent A: 0.1% TFA in H₂O, solvent B: 0.1% TFA in CH₃CN.Product eluted at 30-35% solvent B. Fractions containing pure productwere collected and lyophilized. LC-MS calculated forC₉₇H₁₅₈N₁₅O₃₉S₅[M+H]⁺ 2317.9, found 2318.4.

Synthesis of (Gly)₃-DBCO

The tetrapeptide (Gly)₃-Cys (SEQ ID NO: 12) was synthesized by standardsolid phase peptide synthesis and was dissolved in 0.05 M NaHCO₃ bufferpH 8.3. Maleimide-DBCO (from Click Chemistry Tools) was dissolved inDMSO and then was added to the solution and left to stir at roomtemperature for 1 h until LC-MS indicated near-complete conversion tothe product. The solution was filtered and purified by reversephase-HPLC with a semi-preparative column (Phenomenex, C₁₈ column,Gemini, 5 μm, 10×250 mm) at a flow rate of 5.0 mL/min.; solvent A: 0.1%TFA in H₂O, solvent B: 0.1% TFA in CH₃CN. Product eluted at 35-40%solvent B. Fractions containing pure product were collected andlyophilized. LC-MS calculated for C₄₅H₆₀N₉O₁₃S [M+H]⁺ 966.4, found966.4.

Enzymatic Incorporation of Substrates into Proteins Using Sortase.

The penta-mutant sortase A, with an improved k_(cat), was used (1).Reaction mixtures (1 mL) contained Tris.HCl (50 mM, pH 7.5), CaCl₂ (10mM), NaCl (150 mM), triglycine-containing probe (500 μM),LPETG-containing (SEQ ID NO: 11) substrate (100 μM), and sortase (5 μM)(2, 3). After incubation at 4° C. with agitation for 2 h, reactionproducts were analyzed by LC-MS. Yields were generally >90%. When theyield was below 90%, the reaction was allowed to proceed for anadditional two hours, with addition of sortase to 10 μM andtriglycine-containing probe to 1 mM. Ni-NTA beads were added to thereaction mixture with agitation for 5 min at 25° C. followed bycentrifugation to remove sortase and any remaining unreacted His-taggedsubstrate. The final product was purified by size exclusionchromatography in PBS or Tris.HCl (50 mM, pH 7.5). The labeled proteinwas stored at −80° C. with 5% glycerol for up to six months.

Dimerization of VHHs.

The general procedure was as follows: the DBCO-VHH (1.3 eq, in PBS) wasadded to the azide-X-VHH (where X is either TCO, Texas Red or Alexa647)and the reaction was left to proceed at room temperature for ˜1-3 hourswith constant agitation, where LC-MS analysis revealed (generally) above80% conversion to the corresponding dimer. The dimer was then purifiedvia size exclusion chromatography (FPLC) using PBS as the elutingsolvent. The labeled dimer was stored at −80 OC with 5% glycerol for upto six months.

PEGylation of VHHs.

The general procedure was as follows: the DBCO-PEG (4 eq, in PBS) wasadded to the azide-X-VHH (where X is either TCO, Texas Red or Alexa647)and the reaction was left to proceed at room temperature for ˜1-2 hourswith constant agitation, where SDS-PAGE analysis revealed (generally)above 80% conversion to the corresponding PEGylated product. The finalPEGylated product was purified via size exclusion chromatography (FPLC)using PBS as the eluting solvent. The labeled PEGylated protein wasstored at −80° C. with 5% glycerol for up to six months.

Two-Photon Imaging.

Two-photon imaging was performed with Olympus BX61 upright microscope(Olympus 25×1.05 NA Plan Objective), fitted with a SpectraPysics MaiTaiDeepSee laser. Images were acquired using 910 nm excitation andfollowing filters; CFP (460-510), GFP (495-540) and a third filter(575-630) for the Texas Red signal. Second harmonics (collagen) werealso detected in the CFP filter. Images were acquired with 5 amZ-resolution with Olympus FluoView FC1000 software. Tile images weresaved as JPEG files. Images were processed to obtain a scale bar inImaris v 7.4.0; no intensity or contrast adjustments were made.

Synthesis and Characterization of [¹⁹F]SFB-Tetrazine.

tert-butyl (4-cyanobenzyl)carbamate (2): 4-(Aminomethyl)benzonitrilehydrochloride 1 (2.82 g, 16.7 mmol) and triethylamine (4.7 mL, 33.7mmol) were dissolved in anhydrous CH₂Cl₂ (50 mL) at 0° C. To thisstirred solution was added di-tert-butyl dicarbonate (4.38 g, 20.1mmol), and the reaction allowed to warm to room temperature and stirredfor 16 hours. The reaction mixture was evaporated in vacuo, and theresidue was re-dissolved in diethyl ether (50 mL), which was washedsuccessively with 0.5 M aq. HCl (2×25 mL), saturated NaHCO₃ (2×25 mL)and brine (25 mL). The organic layer was dried with MgSO₄, filtered andevaporated in vacuo to give an off-white solid. The residue was purifiedby flash column chromatography (Hexanes/EtOAc=10/1) to afford tert-butyl(4-cyanobenzyl) carbamate 2 (3.69 g, yield 95%) as a colorless solid. Itwas further characterized according to the literature procedure (4).FIGS. 13A-13B show ¹H-NMR and ¹³C-NMR spectra, respectively.

tert-butyl (4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl) carbamate (3)

A stirred mixture of carbamate 2 (1.5 g, 6.46 mmol), MeCN (3.4 mL, 64.6mmol) and anhydrous NiCl₂ (418 mg, 3.23 mmol) was treated dropwise withhydrazine (5 mL, 161.5 mmol). The purple reaction mixture was stirred at60° C. for 24 hours. Afterwards a solution of NaNO₂ (8.8 g, 127 mmol) inH₂O (65 mL) was carefully added. HCl (2 N solution) was added until theevolution of nitrous oxides ceased. The dark red solution was extractedwith ethyl acetate (3×60 mL). The extract was combined and dried overMgSO₄ and concentrated. The residue was purified by columnchromatography (Hexanes/EtOAc=8/1) to afford tert-butyl(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl) carbamate 3 (1.22 g, yield63%) as a red solid. It was further characterized according to theliterature (5).

(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine (4)

In a 100 mL reaction vessel was charged the solution of tetrazine 3 (301mg, 1.0 mmol) in DCM (12 mL). Trifluoroacetic acid (12 mL) was addeddropwise. The mixture was stirred at room temperature for 2 h.Afterwards the mixture was evaporated and suspended into Et₂O (20 mL)for recrystallization at −20° C. The supernatant was decanted and theresidue was dried under vacuum for 2 hours to afford(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine 4 (200 mg, yield99%) as red solid. The product was further characterized according tothe literature (6).

4-fluoro-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)benzamide (5)

A solution of the tetrazine amine TFA salt 4 (50 mg, 0.25 mmol) inanhydrous DMF (3.5 mL) was added 2,5-dioxopyrrolidin-1-yl4-fluorobenzoate (50 mg, 0.223 mmol) and Et₃N (0.35 mL, 2.5 mmol). Theresulting solution was then stirred at room temperature overnight underargon gas. The reaction mixture was quenched with water (15 mL), andthen extracted with ethyl ether (10 mL×3). The organic layers werecombined, washed with brine, dried over MgSO₄, filtered and evaporatedunder reduced pressure. The crude product was purified by flashchromatography (Hexanes/EtOAc=7/1) to afford4-fluoro-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)benzamide 5 (29 mg,36%) as a red solid powder.

¹H NMR (300 MHz, DMSO) δ 9.18 (t, J=6.1 Hz, 1H), 8.43 (d, J=8.4 Hz, 2H),7.99 (dd, J=5.5, 3.3 Hz, 2H), 7.58 (d, J=8.2 Hz, 2H), 7.31 (t, J=7.8 Hz,2H), 4.60 (d, J=6.1 Hz, 2H), 2.97 (s, 3H); ¹³C NMR (75 MHz, DMSO) δ167.5, 165.9 (d, J=23.3 Hz), 163.6, 162.8, 144.8, 131.0 (d, J=3.1 Hz),130.8, 130.3 (d, J=8.9 Hz), 128.5, 127.9, 115.7 (d, J=21.7 Hz), 43.0,21.3.; HRMS calc'd for C₁₇H₁₅FN₅O⁺ [M+H]⁺, 324.1261; found 324.1265.

Radiochemical Synthesis of [¹⁸F]-Tetrazine ([¹⁸F]-5)

General methods for radioisotope production: a GE PETtrace 16.5 MeVcyclotron was used for [¹⁸F]fluoride production by the ¹⁸O(p,n)¹⁸Fnuclear reaction to irradiate ¹⁸O-enriched water. [¹⁸F]fluoride wasdelivered to a lead-shielded hot cell in ¹⁸O-enriched water by nitrogengas pressure.

General methods for analysis of radiofluorination reactions:radioactivity was quantified using a Capintec Radioisotope Calibrator(CRC-712M) ion chamber. Radiochemical incorporation yields weredetermined by radioTLC. EMD TLC Silica gel 60 plates (10×2 cm) werespotted with an aliquot (1-5 μL) of crude reaction mixture approximately1.5 cm from the bottom of the plate (baseline). TLC plates weredeveloped in a chamber containing ethyl acetate until within 2 cm of thetop of the plate (front). Analysis was performed using a Bioscan AR-2000radio-TLC imaging scanner and WinScan software. Radiochemical identityand purity were determined by radioHPLC. A Phenomenex Luna C18, 250×4.6mm, 5 m HPLC column was used with a Waters 1515 Isocratic HPLC Pumpequipped with a Waters 2487 Dual λ Absorbance Detector, a BioscanFlow-Count equipped with a NaI crystal, and Breeze software.

Manual Radiolabeling.

[¹⁸F]Fluoride was prepared for radiofluorination by the followingmethod: a solution of base (tetraethylammonium bicarbonate (TEAB), 6 mg)in acetonitrile and water (1 mL, v/v 7:3) was added to an aliquot oftarget water (≤1 mL) containing the appropriate amount of [¹⁸F]fluoridein a V-shaped vial sealed with a teflon-lined septum. The vial washeated to 110° C. while nitrogen gas was passed through a P₂O₅-Drierite™column followed by the vented vial. When no liquid was visible in thevial, it was removed from heat, anhydrous acetonitrile (1 mL) was added,and the heating was resumed until dryness. This step was repeated anadditional three times. The vial was then cooled at room temperatureunder nitrogen pressure. The contents were resolubilized in CH₃CN (0.6mL). A solution of TEA[¹⁸F] (0.2 mL) was added into another V-shapedvial charged with 1,4-dinitrobenzene 6 (2 mg) and CH₃CN (0.2 mL). Themixture was heated at 90° C. for 5 min, and then quenched with HPLCmobile phase (40% CH₃CN, 60% 0.1 M NH₄.HCO₂(aq), 0.2 mL). TLC plate wasspotted with crude mixture (2 μL) and developed with 100% EtOAc todetermine the radiochemical conversion. The RCC value (92%) revealedthat the TEA[¹⁸F] solution was ready for radiolabeling. (total time: 8min)

Note:

The quality of TEA[¹⁸F] is crucial for the following radiolabeling, thusthe control is necessary to evaluate the quality.

Ethyl 4-(trimethylammonium triflate)benzoate 8 (4.8 mg) in anhydrousMeCN (0.6 mL) was added to the above dried TEA[¹⁸F] solution (0.4 mL)and the mixture was heated at 90° C. for 10 min to produce ethyl4-[¹⁸F]fluorobenzoate 9. The ethyl ester was subsequently hydrolyzed toform 10 using tetrapropylammonium hydroxide (20 μL, 1.0 M in water) at120° C. for 3 min, and then the mixture azeotropically dried using MeCN(1 mL). Subsequently, a solution ofN,N,N′,N′-Tetramethyl-O—(N-succinimidyl) uronium tetrafluoroborate (10mg) in DMF (0.3 mL) was added and the solution heated at 90° C. for 5min. The mixture was cooled down to ambient temperature. Afterwards asolution of tetrazine amine TFA salt 4 (1.7 mg) in DMF (0.3 mL) wasadded into the mixture, followed by addition of Et₃N (20 μL). Then thereaction was heated at 60° C. for 7 min, quenched with HPLC mobile phase(60% CH₃CN, 40% 0.1 M NH₄—HCO₂(aq), 2 mL). The solution was diluted withwater (15 mL), passed through C18 cartridge, washed with water (10 mL),and eluted with acetonitrile (1.5 mL) to determine the radiochemicalyield (RCY) and identity via co-injection with standard [¹⁹F]-5. FIG. 8shows a table of radiochemical yield (non-decay corrected) and aradioHPLC chromatogram. The column was luna 5u C18 100 Å 250×4.6 mm, themobile phase was 60% CH3CN, 40% 0.1 M NH4.HCO2(aq), and the flow ratewas 1 mL/min.

Automated Synthesis by GE TracerLab FX_(FN) Method.

Following completion of bombardment, the [¹⁸F]fluoride was transferredto the GE TRACERlab™ FX_(FN) radiosynthesis module via helium gasoverpressure. A schematic diagram of the GE medical systems commercialTRACERlab™ FX_(FN) radiosynthesis module used for the synthesis of[¹⁸F]-5 is shown in FIG. 9.

Automated synthesis involves the following: (1) azeotropic drying of[¹⁸F]fluoride; (2) [¹⁸F]fluorination; and (3) HPLC purification,followed by solid-phase formulation of the final product. The synthesismodule was operated in the following sequences with numerical referencesto FIG. 9.

-   -   [¹⁸F]Fluoride was produced by the ¹⁸O(p,n)¹⁸F nuclear reaction        using a GE cyclotron and delivered to the radiosynthesis module        via 10. The [¹⁸F]fluoride was quantitatively trapped on a QMA        carbonate ion exchange solid phase extraction (SPE) light        cartridge (Waters; activated with 6 mL of trace grade H₂O).    -   Automated synthesis began with the elution of resin-bound        [¹⁸F]fluoride using a solution of tetraethylammonium bicarbonate        (6 mg in 500 μL H₂O and 500 μL CH₃CN), pre-loaded into 1 and        delivered to the reactor (12).    -   The reaction mixture (12) was dried azeotropically at 85° C.        under N₂ flow and vacuum over 5 min, then at 110° C. under N₂        flow and vacuum for 2 min, then cooled down to 90° C.    -   Ethyl 4-(trimethylammonium triflate)benzoate 8 (5 mg in 1.0 mL        CH₃CN) pre-loaded into 3 was added to 12. The reactor was sealed        via the closure of valve V13, V20 and V24 and the reaction        mixture was maintained at 90° C. for 10 min.    -   The reaction mixture was then cooled to 40° C., vented via valve        V24, and tetrapropylammonium hydroxide (1.0 M in water, 20 μL in        0.5 mL CH₃CN) pre-loaded into 4 was added to 12. The reactor was        sealed via the closure of valve V24 and the reaction mixture was        heated to 120° C. and this temperature was maintained for 3 min,        then cooled down to 70° C.    -   The reaction mixture (12) was dried azeotropically by addition        of 1 mL anhydrous CH₃CN, preloaded into 5, at 70° C. under N₂        flow and vacuum over 6 min.    -   N,N,N′,N′-Tetramethyl-O—(N-succinimidyl) uronium        tetrafluoroborate (TSTU, 10 mg) in DMF (0.5 mL) pre-loaded into        6 was added to 12. The reactor was sealed via the closure of        valve V24 and the reaction mixture was heated to 90° C. and this        temperature was maintained for 5 min, then cooled down to 60° C.    -   A mixture of tetrazine amine TFA salt 4 (6.0 mg) and Et₃N (40        μL) in DMF (0.5 mL) pre-loaded into 2 was added to 12. The        reaction mixture was maintained at 60° C. for 7 min.    -   The crude reaction mixture was eluted into 14, which was        preloaded with 20:80 CH₃CN/0.1 M ammonium formate solution (3        mL). The contents of 14 were transferred to the HPLC loop via N₂        pressure using a fluid detector, injected onto a        semi-preparative column (Luna C18 semi-preparative, 250×10.00        mm, 5′), and eluted with 40:60 CH₃CN/0.1 M ammonium formate by        volume at a flow rate of 5 mL/min. The eluent was monitored by        UV (X=254 nm) and radiochemical detectors connected in series.    -   A typical semi-preparative HPLC chromatogram is shown in        FIG. 10. The fraction containing the major radiochemical product        (t_(R)=20.1 min) was collected, via valve 18, into a large        dilution vessel (15), which was preloaded with 23 mL of sterile        water for injection (United States Pharmacopeia (USP); Hospira).    -   The diluted HPLC fraction was then loaded onto a C18 SPE        cartridge (16) (Waters; preactivated with 5 mL EtOH followed by        10 mL H₂O).    -   Cartridge 16 was washed with 10 mL sterile water for injection,        USP, preloaded into 7, to remove traces of salts, HPLC mobile        phase, and [¹⁸F]fluoride. Then 16 was eluted with 1.5 mL CH₃CN,        preloaded in 8, into collection vial 17.

Analyses of radioactive mixtures were performed by HPLC with an in-lineUV (X=254 nm) detector in series with a CsI PIN diode radioactivitydetector. Uncorrected radiochemical yields of [¹⁸F]-5 were 10±3% (n=8)relative to starting [¹⁸F]fluoride.

Synthesis and Characterization of [¹⁸F]-VHHs.

General procedure: the solution of [¹⁸F]-Tetrazine 5 obtained fromFX_(FN) was concentrated at 70° C. under N₂ flow for 10 min, then cooleddown to room temperature. A centrifuge tube (1.5 mL) was loaded with PBS(150 μL) and a solution of [¹⁸F]-Tetrazine 5 in CH₃CN (50 μL), then theradioactivity was measured by a dose calibrator (10-15 mCi). VHH-TCO(either monomer or dimer) in PBS (100 μL) was added into the centrifugetube at the last step. The reaction was allowed to proceed with constantagitation at room temperature for ˜20 min. The mixture was analyzed byradio-TLC (100% EtOAc, R_(f)[¹⁸F]-Tz 5=0.6, R_(f) [¹⁸F]-VHHs=0.0)showing more than 80% radiochemical conversion. The reaction mixture wasloaded onto a PD-10 size-exclusion cartridge (GE Healthcare), and PBS(2×500 μL) was used to assist transfer. Afterwards the activity of thereaction centrifuge tube was measured by the dose calibrator (<50 μCi),confirming a complete transfer. The PD-10 cartridge was eluted with PBS(10×500 μL), and each fragment was collected into a new 1.5 mL tube. Thedesired product [¹⁸F]-VHHs usually eluted at tubes #4-7.Characterization (using [¹⁸F]-DC8-dimer as an example): rTLCchromatography (FIG. 11 (left panel) [¹⁸F]-Tz 5; FIG. 11 (right)[¹⁸F]-DC8-dimer; at 20 min). Fragments collection through PD-10cartridge can be found in FIG. 12. After size-exclusion chromatography,a 47±9% (n=5, non-decay corrected) radiochemical yield was obtained.

MicroPET Imaging Studies.

All procedures and animal protocols were approved by the MassachusettsGeneral Hospital subcommittee on research animal care. [¹⁸F]VHHs (20-40μCi) was injected into the tail-vein of each animal. Mice were seriallyimaged using a microPET (Sofie, G4-PET). For all imaging experiments,mice were anesthetized using 2% isoflurane in O₂ at a flow rate of ˜1.5L/min, positioned in a prone position along the long axis of themicroPET scanner and imaged. Images were reconstructed using a filteredback projection reconstruction algorithm. For image analysis,cylindrical regions of interest (ROIs) were manually drawn from threedimensional filtered back projection (FBP) reconstructed PET imagesusing AMIDE software. Regional radioactivity was expressed as thepercentage standardized uptake value [% SUV=% ID/mL×body weight (g)].Two- and three-dimensional visualizations were produced using the DICOMviewer OsiriX (© Pixmeo SARL, 2003-2014).

Sequences of DC8, and DC13 DC8:

Nucleic Acid:

(SEQ ID NO: 1) CAGGTGCAGCTGCAGGAGTCAGGGGGAGGATTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGATTCACATTCAGTACTTACTACATGAGCTGGGTCCGCAAGGCTCCAGGGAAGGGGCCCGAGTGGGTCTCAGTTATGAATAGTAGTGGTGGTGACACAAGGTATGCAGACTTCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACACTGTATCTCCAAATGAACAGCCTGAAACCTGAGGATACGGCCCTGTATTACTGTGCGCAAGGTAGATCAGATATATACCCAACCTTCACGCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGGAGGACTGCCGGAAACCGGC

Peptide:

(SEQ ID NO: 2) QVQLQESGGGLVQPGGSLRLSCTASGFTFSTYYMSWVRKAPGKGPEWVSVMNSSGGDTRYADFVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCAQGRSDIYPTFTRGQGTQVTVSSGSLPETGGHHHHHH

DC13:

Nucleic Acid:

(SEQ ID NO: 3) CAGGTTCAACTGCAAGAGAGTGGCGGGGGCCTGGTTCAGACCGGTGGTTCTCTCCGGCTCTCGTGTGCCGCAAGTGGAGTAGATTTTAACTGGTATAGCATGGGTTGGTTCAGGCAAGCCCCTGGCAAAGAGCGGGAGTATGTGGCTTCGATTGACCAGGGAGGCGAGTTGGATTACGCAATATCAGTAAAGGGCAGATTCACGATCTCCCGAGACAACGCGAAGAATATGGTGTATCTCCAGATGAATTCGTTAAAGCCCGAAGACACCGCTGTATACTACTGTGCCGCAGATTTTTCCGGCCGGGGTGCGTCAAACCCTGACAAGTATAAATATTGGGGACAGGGAACCCAAGTGACCGTCAGCAGCGGTGGGTTGCCCGAAACTGGAGGACACCATC ACCATCACCAT

Peptide:

(SEQ ID NO: 4) QVQLQESGGGLVQTGGSLRLSCAASGVDFNWYSMGWFRQAPGKEREYVASIDQGGELDYAISVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAADFSGRGASNPDKYKYWGQGTQVTVSSGGLPETGGHHHHHH

REFERENCES

-   1. Chen I, Dorr B M, Liu D R (2011) A general strategy for the    evolution of bond-forming enzymes using yeast display. Proc Natl    Acad Sci USA 108(28): 11399-11404.-   2. Theile C S, et al. (2013) Site-specific N-terminal labeling of    proteins using sortase-mediated reactions. Nat Protoc    8(9):1800-1807.-   3. Witte M D, et al. (2012) Preparation of unnatural N-to-N and    C-to-C protein fusions. Proc Natl Acad Sci 109(30): 11993-11998.-   4. Mok N Y, Chadwick J, Kellett K A B, Casas-Arce E, Hooper N M,    Johnson A P, Fishwick C W G (2013) Discovery of    biphenylacetamide-derived inhibitors of BACE1 using de novo    structure-based molecular design. J Med Chem 56(5):1843-1852.-   5. Yang J, Karver M R, Li W, Sahu S, Devaraj N K (2012)    Metal-catalyzed one-pot synthesis of tetrazines directly from    aliphatic nitriles and hydrazine. Angew Chem Int Ed 51(21):    5222-5225.-   6. Evans H L, Carroll L, Aboagye E O, Spivey A C (2014)    Bioorthogonal chemistry for 68Ga radiolabelling of DOTA-containing    compounds. J Label Compd Radiopharm 57(4):291-297.

Example 3. Immuno-PET of Tumor Cells In Vivo

Immunotherapy using checkpoint-blocking antibodies against targets suchas CTLA4 and PD-1 can be used to treat melanoma and non-small cell lungcancer in a subset of patients. The presence of T cells in a tumorcorrelates with improved survival. Immuno-positron emission tomography(immunoPET) was shown to visualize tumors by detecting infiltratinglymphocytes, which can be used to distinguish subjects that respond tocheckpoint inhibitors from subjects that do not respond, or respondpoorly, to checkpoint inhibitors. ⁸⁹Zr-labeled PEGylated single domainantibody fragments (VHHs) specific for the T cell marker MHC II wereused to track the presence of intratumoral T cells by immunoPET.Exemplary methods and compositions for generating such labels are shownin FIGS. 15A and 15B. Such ⁸⁹Zr-labeled PEGylated single domain antibodyfragments may be used to detect/assess tumor infiltrating lymphocytes,e.g., in response to checkpoint blockade in a subject (e.g., a patient),or a biological model of tumor progression (e.g., a B16 melanoma model).⁸⁹Zr-labeled PEGylated-anti-MHC II VHH (VHH7) detected thymus andsecondary lymphoid structures as well as intratumoral MHC II positive Tcells (FIGS. 16A and 16B). Wild-type B6 mice were injected with 0.5million B16 melanoma cells (FIG. 16A) and injected with ⁸⁹Zr-labeledPEGylated-anti-MHC II VHH 7 days following injection of the melanomacells. PEG was 20 kDa in size. PET images were acquired 24 hours postinjection with the ⁸⁹Zr-labeled PEGylated-anti-MHC II VHH. The notationshown in FIG. 16A refers to the following: C-LNs: cervical lymph nodes;Ax: axillary, Br: brachial; KD: kidneys; SP: spleen; BM: bone marrow. Asa control, wild-type B6 mice that were not injected with B6 cells wereinjected with ⁸⁹Zr-labeled PEGylated-anti-MHC II VHH (FIG. 16B). PEG was20 kDa in size. PET images were acquired 24 hours post injection withthe ⁸⁹Zr-labeled PEGylated-anti-MHC II VHH. The notation shown in FIG.16B refers to the following: C-LNs: cervical lymph nodes; Ax: axillary,Br: brachial; KD: kidneys; SP: spleen; BM: bone marrow. The⁸⁹Zr-PEGylated-VHH7 was capable of detecting the B6 tumor cells in themouse by detecting infiltrating lymphocytes into the tumor (FIG. 16A).

Immune responses occur in organized lymphoid structures, whereprofessional antigen presenting cells interact with T and B lymphocytesto protect from infectious disease or cancer. Short of relying onsurvival, immune responses—whether harmful or protective—are commonlyassessed by taking blood samples and measuring the levels of circulatinglymphocytes and their products, such as cytokines and immunoglobulins.In humans, access to bone marrow, spleen and lymph nodes requiressurgical interventions such as biopsies or sampling at autopsy, methodsdifficult to apply on a large scale. Similar limitations apply to thesampling of lymphoid organs in live animals, but at least animal modelsafford the possibility of euthanasia and examination at necropsy of anyorgan or tissue of interest. Most of mouse immunology thus relies onmethods that do not provide longitudinal information for individualanimals. The assessment of immune responses over time therefore remainsa challenge. Intravital multi-photon microscopy can show migration of T,B cells and other immunocytes in real time and has clarified ourunderstanding of, for example, the germinal center reaction.Nonetheless, such mapping of the movement of various lymphocyte andother cell subsets requires complicated invasive procedures. Tracking acellular immune response in a living animal over time remains anunsolved challenge.

The field of tumor immunology has made great progress, in particular inthe areas of antibodies used as checkpoint blockade and for cell-basedtherapies such as chimeric antigen receptor-expressing T cells (CAR-Tcells). For certain cancers (melanoma, non-small cell lung cancer, acutelymphoblastic leukemia) immunotherapy has revolutionized clinicaltreatment and even produced cures, but the failure of a significantfraction of patients to respond—even in these treatable types ofcancer—remains an issue. To follow and visualize immune responseslongitudinally for prognosis would thus be highly desirable.

If it were possible to stratify patients into those who might benefitfrom certain forms of immunotherapy and separate them from those lesslikely to do so, expensive treatments with potentially severe sideeffects might be allocated to those individuals with a high probabilityof response. Positron emission tomography (PET) using labeled antibodiesor antibody fragments (immuno-PET) may achieve this goal.

To achieve the goal of non-invasively monitoring the distribution of Tcells, a small molecular weight antibody-derived format that retainsantigen-binding capability, the variable region segment of camelid heavychain-only antibodies, also referred to as VHHs or nanobodies, wereused. These fragments are typically about 14 kDa in size and readilylend themselves to sortase-catalyzed enzymatic transformations for avariety of purposes, including the installation of radioisotopes for PETimaging.

EQUIVALENTS AND SCOPE

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. It is alsonoted that the terms “comprising” and “containing” are intended to beopen and permits the inclusion of additional elements or steps. Whereranges are given, endpoints are included. Furthermore, unless otherwiseindicated or otherwise evident from the context and understanding of oneof ordinary skill in the art, values that are expressed as ranges canassume any specific value or sub-range within the stated ranges indifferent embodiments of the invention, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the invention can be excluded from any claim,for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

What is claimed is:
 1. A method for site-specifically conjugating ahydrophilic polymer to a single domain antibody comprising: contactingthe single domain antibody comprising a sortase recognition sequencewith a sortase substrate, wherein the sortase substrate is bound to thehydrophilic polymer, in the presence of a sortase to yield asite-specifically modified single domain antibody.
 2. A method for sitespecifically conjugating a hydrophilic polymer to a single domainantibody comprising: contacting the single domain antibody comprising afirst click chemistry handle with a hydrophilic polymer, wherein thehydrophilic polymer comprises a second click chemistry handle, underconditions suitable to yield a single domain antibody conjugate to thehydrophilic polymer.
 3. A method for site-specifically conjugating ahydrophilic polymer to a single domain antibody comprising: (i)contacting a single domain antibody comprising a sortase recognitionsequence with a sortase substrate, wherein the sortase substratecomprises a first click chemistry handle, in the presence of a sortaseto yield a site-specifically modified single domain antibody; and (ii)contacting the site-specifically modified single domain antibody of step(i) with a hydrophilic polymer conjugated to a second click chemistryhandle under conditions suitable to yield a single domain antibodyconjugated to the hydrophilic polymer. 3 a. The method of any one ofclaims 1-3, wherein the hydrophilic polymer is a synthetic polymer. 3 b.The method of any one of claim 1-3, wherein the hydrophilic polymer isnot a polypeptide or polynucleotide.
 4. The method of any one of claims1-3 b, wherein the a hydrophilic polymer is selected from the groupconsisting of polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP),polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyacrylamide,N-(2-hydroxypropyl) methacrylamide (HPMA), divinyl ether-maleicanhydride (DIVEMA), polyoxazoline, polyphosphoester (PPE),polyethyleneimine (PEI), and polyphosphazene.
 5. The method of any oneof claims 1-4, wherein the hydrophilic polymer is polyethylene glycol(PEG), or polyoxazoline.
 6. The method of any one of claims 1-5, whereinthe hydrophilic polymer is polyethylene glycol (PEG).
 7. The method ofany one of claims 1-6, wherein the molecular weight of the hydrophilicpolymer ranges from 2 kDa to 50 kDa.
 8. The method of any one of claims1-7, wherein the molecular weight of the hydrophilic polymer ranges from5 kDa to 40 kDa.
 9. The method of any one of claims 1-8, wherein themolecular weight of the hydrophilic polymer ranges from 5 kDa to 30 kDa.10. The method of any one of claims 1-9, wherein the molecular weight ofthe hydrophilic polymer ranges from 10 kDa to 30 kDa.
 11. The method ofany one of claims 1-10, wherein the molecular weight of the hydrophilicpolymer ranges from 15 kDa to 25 kDa.
 12. The method of any one ofclaims 1-11, wherein the molecular weight of the hydrophilic polymer isapproximately 20 kDa. 12 a. The method of claim 6, wherein the PEGranges from 2 kDa to 50 kDa, 5 kDa to 40 kDa, 5 kDa to 30 kDa, 10 kDa to30 kDa, 15 kDa to 25 kDa, or 15 kDa to 25 kDa.
 13. A method forproducing a site-specifically conjugated bivalent single domain antibodycomprising contacting: a first single domain antibody comprising a firstclick chemistry handle, with a second single domain antibody comprisinga second click chemistry handle, under suitable conditions to yield thesite-specifically conjugated bivalent single domain antibody.
 14. Themethod of claim 13, wherein the first click chemistry handle isconjugated to the C-terminal amino acid residue of the first singledomain antibody.
 15. The method of claim 13 or 14, wherein the secondclick chemistry handle is conjugated to the C-terminal amino acidresidue of the second single domain antibody.
 16. A method for producinga site-specifically conjugated bivalent single domain antibodycomprising: (i) contacting a first single domain antibody comprising asortase recognition sequence with a first sortase substrate, wherein thefirst sortase substrate comprises a first click chemistry handle, in thepresence of a sortase to yield a first site-specifically modified singledomain antibody; (ii) contacting a second single domain antibodycomprising a sortase recognition sequence with a second sortasesubstrate, wherein the second sortase substrate comprises a second clickchemistry handle, in the presence of a sortase, to yield a secondsite-specifically modified single domain antibody; and (iii) contactingthe first site-specifically modified single domain antibody of step (i)with the second site-specifically modified single domain antibody ofstep (ii) under suitable conditions to yield the site-specificallyconjugated bivalent single domain antibody.
 17. The method of claim 16,wherein the first and/or second sortase substrate further comprises adetectable label.
 18. The method of any one of claims 2-17, wherein thefirst click chemistry handle comprises any one of the click chemistryhandles in Table 1 or Table
 2. 19. The method of any one of claims 2-18,wherein the first click chemistry handle comprises a conjugated diene,an optionally substituted tetrazine, an optionally substituted alkene,an optionally substituted trans-cyclooctene (TCO), an aldehyde, aketone, a hydrazine, or an aminooxy functionality.
 20. The method of anyone of claims 2-19, wherein the second click chemistry handle comprisesany one of the click chemistry handles in Table 1 or Table
 2. 21. Themethod of any one of claims 2-20, wherein the second click chemistryhandle comprises a conjugated diene, an optionally substitutedtetrazine, an optionally substituted alkene, an optionally substitutedtrans-cyclooctene (TCO), an aldehyde, a ketone, a hydrazine, or anaminooxy functionality.
 22. The method of any one of claims 2-21,wherein the second click chemistry handle is DBCO.
 23. The method of anyone of claims 2-22, wherein the first click chemistry handle is compound1, 2, 3, or 4 from FIG. 2B.
 24. The method of any one of claims 2-23,wherein the first and/or the second click chemistry handle comprises afluorophore.
 25. The method of claim 24, wherein the fluorophore isAlexa647 or Texas Red.
 26. The method of any one of claims 1-25, whereinthe first sortase substrate and/or the second sortase substratecomprises a peptide.
 27. The method of claim 26, wherein the peptidecomprises the amino acid sequence GGG.
 28. The method of any one ofclaims 1-25, wherein the sortase substrate comprises an alkylaminegroup.
 29. The method of any one of claims 1-28, wherein the sortasesubstrate comprises a linker.
 30. The method of any one of claims 1-29,wherein the first click chemistry handle, or the first sortase substratecomprises a linker.
 31. The method of any one of claims 1-30, whereinthe second click chemistry handle, or the second sortase substratecomprises a linker.
 32. The method of any one of claims 1-31, whereinthe first click chemistry handle, or the first sortase substratecomprises a radionuclide.
 33. The method of any one of claims 1-32,wherein the second click chemistry handle, or the second sortasesubstrate comprises a radionuclide.
 34. The method of claim 32 or 33,wherein the radionuclide is carbon-11, carbon-14, nitrogen-13,oxygen-15, fluorine-18, rubidium-82, copper-61, copper-62, copper-64,yttrium-86, gallium-68, zirconium-89, or iodine-124.
 35. The method ofany one of claims 32-34, wherein the radionuclide is fluorine-18. 36.The method of any one of claims 32-34, wherein the radionuclide iszirconium-89.
 37. The method of any one of claims 32-34, wherein theradionuclide is bound by a chelating moiety.
 38. The method of claim 37,wherein the chelating moiety is 1,4,7-triazacyclononane-triacetic acid(NOTA), 1,4,7,10-tetraazacyclododecane-tetraacetic acid (DOTA),triazacyclononane-phosphinate (TRAP), or desferrioxamine (DFO).
 39. Themethod of claim 36, wherein the zirconium-89 is bound by adesferrioxamine (DFO) chelating moiety.
 40. The method of any one ofclaims 1-39, wherein the first or second click chemistry handle, orfirst or second sortase substrate comprises ¹⁸F-tetrazine or ¹⁸F-FDGconjugated to tetrazine.
 41. The method of any one of claims 1-40,wherein the single domain antibody is a VHH single domain antibody, or asingle chain Fv fragment (scFv).
 42. The method of claim 41, wherein theVHH is DC8 or DC13
 43. The method of any one of claims 13-42, whereinthe first single domain antibody binds CD11b, Class II MHC, CTLA4, CD8,Cd4, CD19, IL2, IL10, CXCL10, or CXCL5.
 44. The method of any one ofclaims 13-43, wherein the first single domain antibody and the secondsingle domain antibody are directed to bind a different antigen orepitope.
 45. The method of any one of claims 13-43, wherein the firstsingle domain antibody and the second single domain antibody aredirected to bind the same antigen or epitope.
 46. The method of any oneof claims 1-12, wherein the sortase substrate comprises a peptide. 47.The method of claim 46, wherein the peptide is an N-terminal peptide.48. The method of claim 46 or 47, wherein the sortase substratecomprises an oligoglycine or an oligoalanine sequence.
 49. The method ofclaim 48, wherein the oligoglycine or oligoalanine comprises 1-10N-terminal glycine residues or 1-10 N-terminal alanine residues,respectively.
 50. The method of any one of claims 46-49, wherein theN-terminal sortase substrate comprises the sequence GGG.
 51. The methodof any one of claims 1-12, wherein the sortase substrate comprises analkylamine group.
 52. The method of any one of claims 16-45, wherein thefirst sortase substrate and/or the second sortase substrate comprises apeptide.
 53. The method of claim 52, wherein the peptide is anN-terminal peptide.
 54. The method of claim 52 or 53, wherein the firstsortase substrate and/or the second sortase substrate comprises anoligoglycine or an oligoalanine sequence.
 55. The method of claim 54,wherein the oligoglycine and/or the oligoalanine comprises 1-10N-terminal glycine residues or 1-10 N-terminal alanine residues,respectively.
 56. The method of any one of claims 52-55, wherein theN-terminal sortase substrate comprises the sequence GGG.
 57. The methodof any one of claims 16-45, wherein the first sortase substrate and/orthe second sortase substrate comprises an alkylamine group.
 58. Themethod of any one of claims 1-57, wherein the sortase recognitionsequence is selected from the group consisting of LPXTX (SEQ ID NO: 15),NPXTX (SEQ ID NO: 16) and LPXAG (SEQ ID NO: 17), wherein each instanceof X independently represents any amino acid residue.
 59. The method ofany of claims 1-57, wherein the enzyme recognition sequence is LPETG(SEQ ID NO: 11), LPETA (SEQ ID NO: 18), NPQTN (SEQ ID NO: 19), NPKTG(SEQ ID NO: 20), LPSTG (SEQ ID NO: 21), or LPXAG(SEQ ID NO: 17).
 60. Themethod of any of claims 1-59, wherein the sortase is sortase A fromStaphylococcus aureus (SrtAaureus), sortase A from Streptococcuspyogenes (SrtApyogenes), sortase B from S. aureus (SrtBaureus), sortaseB from Bacillus anthracis (SrtBanthracis), or sortase B from Listeriamonocytogenes (SrtBmonocytogenes).
 61. A radiolabeled binding proteincomprising: (i) a single domain antibody, (ii) a hydrophilic polymer,and (iii) a radiolabeled agent.
 62. The radiolabeled binding protein ofclaim 61, wherein the single domain antibody is a VHH or an scFv. 63.The radiolabeled binding protein of claim 61 or 62, wherein the singledomain antibody binds to a tumor cell, a tumor-associated cell, or atumor antigen.
 64. The radiolabeled binding protein of claim 61 or 62,wherein the single domain antibody binds to an immune cell.
 65. Theradiolabeled binding protein of claim 64, wherein the lymphocyte is a Tlymphocyte.
 66. The radiolabeled binding protein of claim 64, whereinthe lymphocyte is a B lymphocyte.
 67. The radiolabeled binding proteinof claim 63, wherein the tumor antigen is selected from the groupconsisting of CA-125, MUC-1, and MAGE.
 68. The radiolabeled bindingprotein of claim 63, wherein the tumor cell, or tumor-associated cell isselected from the group consisting of a melanoma cell, a breast cancercell, and a lung cancer cell.
 69. The radiolabeled binding protein ofclaim 61 or 62, wherein the single domain antibody binds to a marker ofinflammation.
 70. The radiolabeled binding protein of claim 69, whereinthe marker of inflammation is selected from the group consisting ofCD11b, CD11c, CD13, CD15, CD66, CD14, CD64, CD66b, CD18, CD16, CD62L,and CD67.
 71. The radiolabeled binding protein of claim 61 or 62,wherein the single domain antibody binds to a cytokine.
 72. Theradiolabeled binding protein of claim 71, wherein the cytokine is IL2,IL10, CXCL10, CXCL5, (TNF)-α, IL-6, IL-1 beta, IL-8, IL-12, IL-16, orIL-18.
 73. The radiolabeled binding protein of claim 30 or 31, whereinthe single domain antibody binds to CD8, CTLA4, MHC class II, or CD11b,CD4, CD19, IL2, IL10, CXCL10, or CXCL5.
 74. The radiolabeled bindingprotein of any one of claims 61-73, wherein the single domain antibodyis from 10 kDa to 40 kDa in size.
 75. The radiolabeled binding proteinof any one of claims 61-74, wherein the single domain antibody is from10 kDa to 20 kDa in size.
 76. The radiolabeled binding protein of anyone of claims 61-75, wherein the single domain antibody is from 10 kDato 18 kDa in size.
 77. The radiolabeled binding protein of any one ofclaims 61-76, wherein the single domain antibody comprises a sortaserecognition sequence.
 78. The radiolabeled binding protein of any one ofclaims 61-77, wherein the single domain antibody comprises a C-terminalsortase recognition sequence.
 79. The radiolabeled binding protein ofclaim 77 or 78, wherein the sortase recognition sequence is LPXTX (SEQID NO: 15), wherein each instance of X independently represents anyamino acid residue.
 80. The radiolabeled binding protein of claim 77 or78, wherein the sortase recognition sequence is LPETG (SEQ ID NO: 11) orLPETA (SEQ ID NO:18).
 81. The radiolabeled binding protein of claim 77or 78, wherein the sortase recognition motif is NPXTX (SEQ ID NO: 16),wherein each instance of X independently represents any amino acidresidue.
 82. The radiolabeled binding protein of claim 77 or 78, whereinthe sortase recognition motif is NPQTN (SEQ ID NO: 19) or NPKTG (SEQ IDNO: 20). 82 a. The radiolabeled binding protein of any one of claims61-82, wherein the hydrophilic polymer is selected from the groupconsisting of polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP),polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyacrylamide,N-(2-hydroxypropyl) methacrylamide (HPMA), divinyl ether-maleicanhydride (DIVEMA), polyoxazoline, polyphosphoester (PPE),polyethyleneimine (PEI), and polyphosphazene.
 83. The radiolabeledbinding protein of any one of claims 61-82 a, wherein the hydrophilicpolymer is a polyethylene glycol (PEG) or polyoxazoline.
 84. Theradiolabeled binding protein of any one of claims 61-83, wherein thehydrophilic polymer is a polyethylene glycol (PEG).
 85. The radiolabeledbinding protein of any one of claims 61-84, wherein the molecular weightof the hydrophilic polymer ranges from 2 kDa to 50 kDa.
 86. Theradiolabeled binding protein of any one of claims 61-85, wherein themolecular weight of the hydrophilic polymer ranges from 5 kDa to 40 kDa.87. The radiolabeled binding protein of any one of claims 61-86, whereinthe molecular weight of the hydrophilic polymer ranges from 10 kDa to 30kDa.
 88. The radiolabeled binding protein of any one of claims 61-87,wherein the molecular weight of the hydrophilic polymer ranges from 15kDa to 25 kDa.
 89. The radiolabeled binding protein of any one of claims61-88, wherein the molecular weight of the hydrophilic polymer isapproximately 20 kDa.
 90. The radiolabeled binding protein of any one ofclaims 61-89, wherein the radiolabeled agent comprises a radionuclidethat is carbon-11, carbon-14, nitrogen-13, oxygen-15, fluorine-18,rubidium-82, copper-61, copper-62, copper-64, yttrium-86, gallium-68,zirconium-89, or iodine-124.
 91. The radiolabeled binding protein ofclaim 90, wherein the radiolabeled agent comprises a radionuclide thatis zirconium-89, fluorine-18, copper-64, or gallium-68.
 92. Theradiolabeled binding protein of claim 90, wherein the radiolabeled agentcomprises a radionuclide that is zirconium-89.
 93. The radiolabeledbinding protein of claim 90, wherein the radiolabeled agent comprises aradionuclide that is fluorine-18.
 94. The radiolabeled binding proteinof any one of claims 61-90, wherein the radiolabeled agent isfludeoxyglucose (18F-FDG).
 95. The radiolabeled binding protein of anyone of claims 61-90, wherein the radiolabeled agent comprises ¹⁸F. 96.The radiolabeled binding protein of any one of claims 61-95 furthercomprising a chelating moiety.
 97. The radiolabeled binding protein ofclaim 58, wherein the chelating moiety is1,4,7-triazacyclononane-triacetic acid (NOTA),1,4,7,10-tetraazacyclododecane-tetraacetic acid (DOTA), ortriazacyclononane-phosphinate (TRAP), or desferrioxamine (DFO).
 98. Theradiolabeled binding protein of claim 97 or 96, wherein the radionuclideis bound by the chelating moiety.
 99. A single domain antibody producedby the method of any one of claims 1-60.
 100. A method of diagnosing,monitoring, imaging, or treating a subject comprising: (a) administeringthe radiolabeled binding protein of any one of claims 61-98, or thesingle domain antibody of claim 99, to the subject; and (b) detectingthe radiolabel in the subject.
 101. The method of claim 100, wherein thesubject has, has had, or is suspected of having cancer.
 102. The methodof claim 100, wherein the subject has, has had, or is suspected ofhaving an inflammatory disease or disorder.
 103. The method of any oneof claims 100-102, wherein step (b) is performed using positron emissiontomography (PET).
 104. A method of obtaining a radiologic image of asubject, comprising: (i) administering the radiolabeled binding proteinof any one of claims 61-98, or the single domain antibody of claim 99,to the subject; and (ii) obtaining the radiologic image of the subjectby capturing the radiation emitted.
 105. The method of claim 104,wherein a tissue, an organ, or a tumor of the subject is imaged.
 106. Amethod of obtaining a radiologic image of a biological sample,comprising: (i) contacting the biological sample with the radiolabeledbinding protein of any one of claims 61-98, or the single domainantibody of claim 99, to the subject; and (ii) obtaining the radiologicimage of the biological sample by capturing the radiation emitted. 107.The method of claim 106, wherein the biological sample is ex vivo or invitro.
 108. The method of claim 106 or 107, wherein the biologicalsample is a tumor.
 109. A method of treating a subject having a tumor,the method comprising: (i) administering the radiolabeled bindingprotein of any one of claims 61-98, or the single domain antibody ofclaim 99, to the subject; (ii) obtaining a radiologic image of thetumor; (iii) determining an intensity or a pattern of the radiologicimage; (iv) administering an immune checkpoint inhibitor to the subjectbased on the intensity or the pattern of the radiologic image determinedin step (iii).
 110. The method of claim 109, wherein the immunecheckpoint inhibitor is administered to the subject only if theintensity of the radiologic image is above a threshold.
 111. The methodof claim 109, wherein the immune checkpoint inhibitor is administered tothe subject only if the intensity of the radiologic image is greaterthan the intensity of a suitable control.
 112. The method of claim 109,wherein the immune checkpoint inhibitor is administered to the subjectonly if greater than 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the tumoris detected by the radiolabeled binding protein of any one of claims61-98, or the single domain antibody of claim
 99. 113. The method of anyone of claims 109-112, wherein the tumor is from a melanoma, a non-smallcell lung cancer, a kidney cancer, a head and neck cancer, or aHodgkin's lymphoma.
 114. The method of any one of claims 109-113,wherein the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1,CTLA4, B7-1, or B7-2.
 115. The method of any one of claims 109-114,wherein the immune checkpoint inhibitor is an antibody that binds toPD-1, PD-L1, CTLA4, B7-1, or B7-2.
 116. The method of any one of claims109-115, wherein the immune checkpoint inhibitor is ipilimumab,pembrolizumab, atezolizumab, or nivolumab.
 117. The method of any one ofclaims 109-116 further comprising administering a chemotherapeutic agentto the subject.
 118. A composition comprising the radiolabeled bindingprotein of any one of claims 61-98, or the single domain antibody ofclaim 99, and an excipient.
 119. A pharmaceutical composition comprisingthe radiolabeled binding protein of any one of claims 61-98, or thesingle domain antibody of claim 99, and a pharmaceutically acceptablecarrier.
 120. A method of diagnosing, monitoring, imaging, or treating asubject comprising: (a) administering the composition of claim 118 or119 to the subject; and (b) detecting the radiolabel in the subject.121. The method of claim 120, wherein the subject has, has had, or issuspected of having cancer.
 122. The method of claim 120, wherein thesubject has, has had, or is suspected of having an inflammatory diseaseor disorder.
 123. The method of any one of claims 120 or 122, whereinstep (b) is performed using positron emission tomography (PET).
 124. Akit for site-specifically conjugating a hydrophilic polymer to a singledomain antibody comprising: (i) a single domain antibody comprising asortase recognition sequence, and (ii) a sortase substrate, wherein thesortase substrate is bound to the hydrophilic polymer.
 125. The kit ofclaim 124, wherein the kit further comprises a sortase.
 126. A kit forsite-specifically conjugating a hydrophilic polymer to a single domainantibody comprising: (i) a single domain antibody comprising a firstclick chemistry handle, and (ii) a hydrophilic polymer, wherein thehydrophilic polymer is conjugated to a second click chemistry handle.127. A kit for site-specifically conjugating a hydrophilic polymer to asingle domain antibody comprising: (i) a single domain antibodycomprising a sortase recognition sequence, (ii) a sortase substratecomprising a first click chemistry handle, and (iii) a hydrophilicpolymer conjugated to a second click chemistry handle.
 128. The kit ofclaim 127, wherein the kit further comprises a sortase.
 129. A kit forproducing a site-specifically conjugated bivalent single domain antibodycomprising: (i) a first single domain antibody comprising a first clickchemistry handle, and (ii) a second single domain antibody comprising asecond click chemistry handle.
 130. A kit for producing asite-specifically conjugated bivalent single domain antibody comprising:(i) a first single domain antibody comprising a sortase recognitionsequence, (ii) a first sortase substrate, wherein the first sortasesubstrate comprises a first click chemistry handle, (iii) a secondsingle domain antibody comprising a sortase recognition sequence, and(iv) a second sortase substrate, wherein the second sortase substratecomprises a second click chemistry handle.
 131. The kit of claim 130further comprising a sortase.
 132. The kit of any one of claims 124-131further comprising a radionuclide.
 133. The kit of claim 132, whereinthe radionuclide is carbon-11, carbon-14, nitrogen-13, oxygen-15,fluorine-18, rubidium-82, copper-61, copper-62, copper-64, yttrium-86,gallium-68, zirconium-89, or iodine-124.
 134. The kit of any one ofclaims 124-133 further comprising sortase A from Staphylococcus aureus(SrtAaureus), sortase A from Streptococcus pyogenes (SrtApyogenes),sortase B from S. aureus (SrtBaureus), sortase B from Bacillus anthracis(SrtBanthracis), or sortase B from Listeria monocytogenes(SrtBmonocytogenes).